94 human secreted proteins

ABSTRACT

The present invention relates to novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to these novel human secreted proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.10/115,123, filed Apr. 4, 2002, which is a divisional of U.S.application Ser. No. 09/461,325, filed Dec. 14, 1999, now U.S. Pat. No.6,475,753, issued on Nov. 5, 2002, which is a continuation-in-part ofInternational Application No. PCT/US99/13418, filed Jun. 15, 1999, whichclaims benefit under 35 U.S.C. § 119(e) of U.S. Provisional ApplicationNo. 60/089,507, filed Jun. 16, 1998, 60/089,508, filed Jun. 16, 1998,60/089,509, filed Jun. 16, 1998, 60/089,510, filed Jun. 16, 1998,60/090,112, filed Jun. 22, 1998, and 60/090,113, filed Jun. 22, 1998,all of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides andthe polypeptides encoded by these polynucleotides, uses of suchpolynucleotides and polypeptides, and their production.

BACKGROUND OF THE INVENTION

[0003] Unlike bacterium, which exist as a single compartment surroundedby a membrane, human cells and other eucaryotes are subdivided bymembranes into many functionally distinct compartments. Eachmembrane-bounded compartment, or organelle, contains different proteinsessential for the function of the organelle. The cell uses “sortingsignals,” which are amino acid motifs located within the protein, totarget proteins to particular cellular organelles.

[0004] One type of sorting signal, called a signal sequence, a signalpeptide, or a leader sequence, directs a class of proteins to anorganelle called the endoplasmic reticulum (ER). The ER separates themembrane-bounded proteins from all other types of proteins. Oncelocalized to the ER, both groups of proteins can be further directed toanother organelle called the Golgi apparatus. Here, the Golgidistributes the proteins to vesicles, including secretory vesicles, thecell membrane, lysosomes, and the other organelles.

[0005] Proteins targeted to the ER by a signal sequence can be releasedinto the extracellular space as a secreted protein. For example,vesicles containing secreted proteins can fuse with the cell membraneand release their contents into the extracellular space—a process calledexocytosis. Exocytosis can occur constitutively or after receipt of atriggering signal. In the latter case, the proteins are stored insecretory vesicles (or secretory granules) until exocytosis istriggered. Similarly, proteins residing on the cell membrane can also besecreted into the extracellular space by proteolytic cleavage of a“linker” holding the protein to the membrane.

[0006] Despite the great progress made in recent years, only a smallnumber of genes encoding human secreted proteins have been identified.These secreted proteins include the commercially valuable human insulin,interferon, Factor VIII, human growth hormone, tissue plasminogenactivator, and erythropoeitin. Thus, in light of the pervasive role ofsecreted proteins in human physiology, a need exists for identifying andcharacterizing novel human secreted proteins and the genes that encodethem. This knowledge will allow one to detect, to treat, and to preventmedical disorders by using secreted proteins or the genes that encodethem.

SUMMARY OF THE INVENTION

[0007] The present invention relates to novel polynucleotides and theencoded polypeptides. Moreover, the present invention relates tovectors, host cells, antibodies, and recombinant and synthetic methodsfor producing the polypeptides and polynucleotides. Also provided arediagnostic methods for detecting disorders and conditions related to thepolypeptides and polynucleotides, and therapeutic methods for treatingsuch disorders and conditions. The invention further relates toscreening methods for identifying binding partners of the polypeptides.

DETAILED DESCRIPTION

[0008] Definitions

[0009] The following definitions are provided to facilitateunderstanding of certain terms used throughout this specification.

[0010] In the present invention, “isolated” refers to material removedfrom its original environment (e.g., the natural environment if it isnaturally occurring), and thus is altered “by the hand of man” from itsnatural state. For example, an isolated polynucleotide could be part ofa vector or a composition of matter, or could be contained within acell, and still be “isolated” because that vector, composition ofmatter, or particular cell is not the original environment of thepolynucleotide. The term “isolated” does not refer to genomic or cDNAlibraries, whole cell total or mRNA preparations, genomic DNApreparations (including those separated by electrophoresis andtransferred onto blots), sheared whole cell genomic DNA preparations orother compositions where the art demonstrates no distinguishing featuresof the polynucleotide/sequences of the present invention.

[0011] In the present invention, a “secreted” protein refers to thoseproteins capable of being directed to the ER, secretory vesicles, or theextracellular space as a result of a signal sequence, as well as thoseproteins released into the extracellular space without necessarilycontaining a signal sequence. If the secreted protein is released intothe extracellular space, the secreted protein can undergo extracellularprocessing to produce a “mature” protein. Release into the extracellularspace can occur by many mechanisms, including exocytosis and proteolyticcleavage.

[0012] In specific embodiments, the polynucleotides of the invention areat least 15, at least 30, at least 50, at least 100, at least 125, atleast 500, or at least 1000 continuous nucleotides but are less than orequal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotidesof the invention comprise a portion of the coding sequences, asdisclosed herein, but do not comprise all or a portion of any intron. Inanother embodiment, the polynucleotides comprising coding sequences donot contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′to the gene of interest in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1genomic flanking gene(s).

[0013] As used herein, a “polynucleotide” refers to a molecule having anucleic acid sequence contained in SEQ ID NO:X or the cDNA containedwithin the clone deposited with the ATCC. For example, thepolynucleotide can contain the nucleotide sequence of the full lengthcDNA sequence, including the 5′ and 3′ untranslated sequences, thecoding region, with or without the signal sequence, the secreted proteincoding region, as well as fragments, epitopes, domains, and variants ofthe nucleic acid sequence. Moreover, as used herein, a “polypeptide”refers to a molecule having the translated amino acid sequence generatedfrom the polynucleotide as broadly defined.

[0014] In the present invention, the full length sequence identified asSEQ ID NO:X was often generated by overlapping sequences contained inmultiple clones (contig analysis). A representative clone containing allor most of the sequence for SEQ ID NO:X was deposited with the AmericanType Culture Collection (“ATCC”). As shown in Table 1, each clone isidentified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number.The ATCC is located at 10801 University Boulevard, Manassas, Va.20110-2209, USA. The ATCC deposit was made pursuant to the terms of theBudapest Treaty on the international recognition of the deposit ofmicroorganisms for purposes of patent procedure.

[0015] A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:X, the complementthereof, or the cDNA within the clone deposited with the ATCC.“Stringent hybridization conditions” refers to an overnight incubationat 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mMNaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65 degree C.

[0016] Also contemplated are nucleic acid molecules that hybridize tothe polynucleotides of the present invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37 degree C. ina solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA;followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition,to achieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

[0017] Note that variations in the above conditions may be accomplishedthrough the inclusion and/or substitution of alternate blocking reagentsused to suppress background in hybridization experiments. Typicalblocking reagents include Denhardt's reagent, BLOTTO, heparin, denaturedsalmon sperm DNA, and commercially available proprietary formulations.The inclusion of specific blocking reagents may require modification ofthe hybridization conditions described above, due to problems withcompatibility.

[0018] Of course, a polynucleotide which hybridizes only to polyA+sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in thesequence listing), or to a complementary stretch of T (or U) residues,would not be included in the definition of “polynucleotide,” since sucha polynucleotide would hybridize to any nucleic acid molecule containinga poly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer)

[0019] The polynucleotide of the present invention can be composed ofany polyribonucleotide or polydeoxribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. For example,polynucleotides can be composed of single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, the polynucleotidecan be composed of triple-stranded regions comprising RNA or DNA or bothRNA and DNA. A polynucleotide may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

[0020] The polypeptide of the present invention can be composed of aminoacids joined to each other by peptide bonds or modified peptide bonds,i.e., peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

[0021] “SEQ ID NO:X” refers to a polynucleotide sequence while “SEQ IDNO:Y” refers to a polypeptide sequence, both sequences identified by aninteger specified in Table 1.

[0022] “A polypeptide having biological activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical to, anactivity of a polypeptide of the present invention, including matureforms, as measured in a particular biological assay, with or withoutdose dependency. In the case where dose dependency does exist, it neednot be identical to that of the polypeptide, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thepolypeptide of the present invention (i.e., the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about tenfold less activity, and mostpreferably, not more than about three-fold less activity relative to thepolypeptide of the present invention.)

[0023] Polynucleotides and Polypeptides of the Invention

[0024] Features of Protein Encoded by Gene NO: 1

[0025] Preferred polypeptides of the invention comprise the followingamino acid sequence: TRPEKVQAPLKWFKFQILDPP (SEQ ID NO: 253).Polynucleotides encoding these polypeptides are also provided.

[0026] This gene is expressed primarily in dendritic cells and to alesser extent in other tissues.

[0027] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immune,nervous system, and inflammatory disorders. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., immune, cancerous and woundedtissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0028] The tissue distribution in dendritic cells indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the detection/treatment of neurodegenerative disease states andbehavioral disorders such as Alzheimer's Disease, Parkinson's Disease,Huntington's Disease, schizophrenia, mania, dementia, paranoia,obsessive compulsive disorder, panic disorder, and autism. In addition,the gene or gene product may also play a role in the treatment and/ordetection of developmental disorders associated with the developingembryo, sexually-linked disorders, or disorders of the cardiovascularsystem. Furthermore, expression of this gene product in primarydendritic cells also indicates that it may play a role in mediatingresponses to infection and controlling immunological responses, such asthose that occur during immune surveillance. Representative uses aredescribed in the “Immune Activity” and “Infectious Disease” sectionsbelow, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhereherein. Furthermore, the protein may also be used to determinebiological activity, raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker.

[0029] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:11 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 885 of SEQID NO:11, b is an integer of 15 to 899, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:11, and where bis greater than or equal to a +14.

[0030] Features of Protein Encoded by Gene NO: 2

[0031] The translation product of this gene share homology with the Tbc1gene of Mus musculus which is thought to play a role in the cell cycleand differentiation of various tissues (See Genbank accession no.gi|988221 as well as Medline article no.96032578; all referencesavailable through these accessions are hereby incorporated by referenceherein). One embodiment for this gene is the polypeptide fragmentscomprising the following amino acid sequence:SAEFGVAPLPGRRGSPVRQLAQFRRRLLRGSGGRGAPGRPPRCPGEARVMXPPSCIQDEPFPHPLEPEPGVSAQPGPGKPSDKRFRLWYVGGSCLDHRTTLPMLPWLMAEIRRRSQKPEAGGCGAPAAREVILVLSAPFLRCVPAPGAGASGGTSPSATQPNPAVFIFEHKAQHISRFIHNSHDLTYFAYLIKAQPDDPESQMACHVFRATDPSQVPDVISSIRQLSKXAMKEDAKPSKDNEDAFYNSQKFEVLYCGKVTVTP QEGPLKPHR (SEQ IDNO: 254), PMLPWLMAEIRRRS (SEQ ID NO: 255), JHNSHDLTYFAYLIKAQPD (SEQ IDNO: 256), KFEVLYCGKVTV (SEQ ID NO: 257), and/or ISSIRQLSKAMKE (SEQ IDNO: 258). Polynucleotides encoding these polypeptides are also provided.

[0032] This gene is expressed primarily in smooth muscle and dendriticcells and to a lesser extent in other tissues.

[0033] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,cardiovascular diseases and immune and inflammatory disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the immunesystem and cardiovascular system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., smooth muscle and dendritic cells,cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

[0034] The tissue distribution in smooth muscle and dendritic cells andhomology to a protein involved in regulation of cell cycle and tissuedifferentiation indicates that polynucleotides and polypeptidescorresponding to this gene are useful for the detection/treatment and/orprevention of immune system disorders, cardiovascular disorders ordiseases, including cancer and other proliferative disorders.

[0035] The tissue distribution indicates polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosis andtreatment of a variety of immune system disorders. Representative usesare described in the “Immune Activity” and “Infectious Disease” sectionsbelow, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhereherein. Briefly, the expression of this gene product indicates a role inregulating the proliferation; survival; differentiation; and/oractivation of hematopoietic cell lineages, including blood stem cells.

[0036] This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes suggests ausefulness in the treatment of cancer (e.g., by boosting immuneresponses). Expression in cells of lymphoid origin, the natural geneproduct may be involved in immune functions. Therefore it may be alsoused as an agent for immunological disorders including arthritis,asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoidarthritis, granulomatous disease, inflammatory bowel disease, sepsis,acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such asT-cell mediated cytotoxicity; immune reactions to transplanted organsand tissues, such as host-versus-graft and graft-versus-host diseases,or autoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, sclerodermaand tissues.

[0037] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types.Alternatively, the protein is useful in the detection, treatment, and/orprevention of vascular conditions, which include, but are not limitedto, microvascular disease, vascular leak syndrome, aneurysm, stroke,atherosclerosis, arteriosclerosis, or embolism. For example, this geneproduct may represent a soluble factor produced by smooth muscle thatregulates the innervation of organs or regulates the survival ofneighboring neurons. Likewise, it may be involved in controlling thedigestive process, and such actions as peristalsis. Similarly, it may beinvolved in controlling the vasculature in areas where smooth musclesurrounds the endothelium of blood vessels. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0038] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:12 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1126 of SEQID NO:12, b is an integer of 15 to 1140, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:12, and whereb is greater than or equal to a +14.

[0039] Features of Protein Encoded by Gene NO: 3

[0040] The translation product of this gene shares sequence homologywith alpha-I antitrypsin (See Genbank accession no. gnl|PID|d1021080 andBAA20264; all references available through this accession are herebyincorporated by reference herein). Alpha-1-antitrypsin is an importantplasma protease inhibitor affecting a wide variety of serine proteasesinvolved in coagulation, fibrinolysis and kinen generation.

[0041] Preferred polypeptides of the invention comprise the followingamino acid sequence: GERRNWGGEVYYSTGYSSRK (SEQ ID NO: 259).Polynucleotides encoding these polypeptides are also provided. Thepolypeptide of this gene has been determined to have a transmembranedomain at about amino acid position 22-414 of the amino acid sequencereferenced in Table 1 for this gene. Moreover, a cytoplasmic tailencompassing amino acids 5-21 of this protein has also been determined.Based upon these characteristics, it is believed that the proteinproduct of this gene shares structural features to type Ib membraneproteins.

[0042] This gene is expressed primarily in healing groin wound and to alesser extent in some other tissues.

[0043] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, woundhealing disorders. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thehealing groin wound, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., healing, regenerative, cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder. Preferred polypeptides of thepresent invention comprise immunogenic epitopes shown in SEQ ID NO: 134as residues: Phe-25 to Tyr-30, Gln-37 to Arg-42, Lys-106 to Leu-112,Leu-123 to Leu-130, Gln-142 to Phe-150, Gln-183 to Lys-188, Asp-219 toGlu-226, Lys-359 to Glu-366. Polynucleotides encoding said polypeptidesare also provided.

[0044] The tissue distribution in healing groin wound and homology toalpha-1 antitrypsin indicates that polynucleotides and polypeptidescorresponding to this gene are useful for diagnosis and therapeutictreatment of wound healing disorders. In addition, since healing woundshave transcriptional environments similar to developing tissues, thetranslation product of this gene may be useful for the diagnosis andtreatment of cancer and other proliferative disorders. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0045] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:13 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1431 of SEQID NO:13, b is an integer of 15 to 1445, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:13, and whereb is greater than or equal to a +14.

[0046] Features of Protein Encoded by Gene NO: 4

[0047] The translation product of this gene shares homology with membersof the HEMK family of modification methylases (See, e.g., GenbankAccession No. gb|AAD26417.1|AF131220_(—)1; all references availablethrough this accession are hereby incorporated by reference herein).

[0048] Preferred polypeptides of the invention comprise the followingamino acid sequence: EPGAAQESW (SEQ ID NO: 260),LCARPSCSYTGAENQGQPRSPGWGSSHVGWGWGVGSPFLGSQEWSGLAPDLPDQEEEQPVGRHSCPDMSQCIKRGHQPVGFSKHAWRCLVGCCPWEEEKRSCHPFGAXLLWVLRFALQPXVYEDPAALDGGEEGMDIXTHILALAPRLLKDSGSIFLEVDPRHPXLVSSWLQSRPDLYLNLVAVRRDFCGRPRFLHIRRSGP (SEQ ID NO: 261),LCARPSCSYTGAENQGQPRSPGWGSSHVGWGWGVGSP (SEQ ID NO: 262),FLGSQEWSGLAPDLPDQEEEQPVGRHSCPDMSQCIKR (SEQ ID NO: 263),GHQPVGFSKHAWRCLVGCCPWEEEKRSCHPFGAXLLW (SEQ ID NO: 264),VLRFALQPXVYEDPAALDGGEEGMDIXTHILALAPRL (SEQ ID NO: 265), and/orLKDSGSIFLEVDPRHPXLVSSWLQSRPDLYLNLVAVRRDFCGRPRFLHIRRSGP (SEQ ID NO: 266).Polynucleotides encoding these polypeptides are also provided.

[0049] This gene is expressed primarily in immune (e.g., B-cells andneutrophils) and tumor tissues, and to a lesser extent in some othertissues such as heart.

[0050] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immuneand inflammatory disorders and tumorigenesis. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune and tumor tissues, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., cancerous and wounded tissues) orbodily fluids (e.g., serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder. Preferred polypeptides of thepresent invention comprise immunogenic epitopes shown in SEQ ID NO: 135as residues: Met-1 to Cys-6, Ser-26 to Gly-35. Polynucleotides encodingsaid polypeptides are also provided.

[0051] The tissue distribution in tumors of immune origins indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for diagnosis and intervention of such tumors, in addition toother tumors where expression has been indicated. Additionally, thisgene is a good target for antagonists, particularly small molecules orantibodies, which block binding of the receptor by its cognateligand(s).

[0052] The tissue distribution in neutrophils and B-cells indicatespolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Briefly, the expression indicates arole in regulating the proliferation; survival; differentiation; and/oractivation of hematopoietic cell lineages, including blood stem cells.Involvement in the regulation of cytokine production, antigenpresentation, or other processes indicates a usefulness for treatment ofcancer (e.g. by boosting immune responses). Expression in cells oflymphoid origin, indicates the natural gene product would be involved inimmune functions. Therefore it would also be useful as an agent forimmunological disorders including arthritis, asthma, immunodeficiencydiseases such as AIDS, leukemia, rheumatoid arthritis, granulomatousdisease, inflammatory bowel disease, sepsis, acne, neutropenia,neutrophilia, psoriasis, hypersensitivities, such as T-cell mediatedcytotoxicity; immune reactions to transplanted organs and tissues, suchas host-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.

[0053] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. Thus, this gene productis thought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, to raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0054] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:14 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1194 of SEQID NO:14, b is an integer of 15 to 1208, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:14, and whereb is greater than or equal to a +14.

[0055] Features of Protein Encoded by Gene NO: 5

[0056] The translation product of this gene shares sequence homologywith mouse von Ebner minor salivary gland protein which may play a rolein carbohydrate metabolism (See Genbank Accession No. gb|AAA87581.1|;all references available through this accession are hereby incorporatedby reference herein).

[0057] Preferred polypeptides of the invention comprise the followingamino acid sequence: QELLVKIPLDMVAGFNTPL (SEQ ID NO: 267),LRIQLLHKLSFLVNALAKQVMNLLVP (SEQ ID NO: 268),AGPWTFTLLCGLLAATLIQATLSPTAVLILGPKVIKEKLTQELKDHNATSILQQ LPLL (SEQ ID NO:270), and/or HXIWLKVITXNILQLQVKPS (SEQ ID NO: 269). Polynucleotidesencoding these polypeptides are also provided.

[0058] This gene is expressed primarily in respiratory tissues such astrachea, larynx and other pulmonary tissues, and to a lesser extent inother tissues.

[0059] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,respiratory system and oral disorders. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the respiratory tissues, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., cancerous and wounded tissues) orbodily fluids (e.g., serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder. Preferred polypeptides of thepresent invention comprise immunogenic epitopes shown in SEQ ID NO: 136as residues: Lys-39 to Asn-48, Arg-63 to Gly-68, Pro-101 to Gln-106.Polynucleotides encoding said polypeptides are also provided.

[0060] The tissue distribution combined with the homology to von Ebnerminor salivary gland protein indicates that polynucleotides andpolypeptides corresponding to this gene are useful for diagnosis andtreatment of respiratory and oral diseases.

[0061] Furthermore, the tissue distribution in pulmonary tissues alsoindicates that polynucleotides and polypeptides corresponding to thisgene are useful for diagnosis and intervention of tumors within thesetissues, in addition to other tumors where expression has beenindicated. Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tumors and tissues. Protein may show utility in thediagnosis, treatment, and/or prevention of disorders in carbohydratemetabolism.

[0062] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:15 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1161 of SEQID NO:15, b is an integer of 15 to 1175, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:15, and whereb is greater than or equal to a +14.

[0063] Features of Protein Encoded by Gene NO: 6

[0064] The gene encoding the disclosed cDNA is believed to reside onchromosome 2. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 2.

[0065] This gene is expressed primarily in fast-growing tissues such asfetal tissues, hematopoietic cells and tumor tissues and to a lesserextent in other tissues.

[0066] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, growthdisorders, tumorigenesis, and immune or inflammatory disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of thefast-growing tissues such as fetal tissues, hematopoietic cells andtumor tissues, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

[0067] The tissue distribution in fast growing tissues indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of cancer and other proliferativedisorders.

[0068] Expression in embryonic tissue and other cellular sources markedby proliferating cells indicates that this protein may play a role inthe regulation or cellular division. Additionally, the expression inhematopoietic cells and tissues indicates that this protein may play arole in the proliferation, differentiation, and/or survival ofhematopoietic cell lineages which implicates the protein product of thisgene as being useful for the treatment and diagnosis of hematopoeticrelated disorders such as anemia, pancytopenia, leukopenia,thrombocytopenia or leukemia since stromal cells are important in theproduction of cells of hematopoietic lineages. Representative uses aredescribed in the “Immune Activity” and “Infectious Disease” sectionsbelow, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhereherein. Briefly, the uses include bone marrow cell ex vivo culture, bonemarrow transplantation, bone marrow reconstitution, radiotherapy orchemotherapy of neoplasia.

[0069] The gene product may also be involved in lymphopoiesis,therefore, it can be used in immune disorders such as infection,inflammation, allergy, immunodeficiency etc. Thus, this gene may beuseful in the treatment of lymphoproliferative disorders, and in themaintenance and differentiation of various hematopoietic lineages fromearly hematopoietic stem and committed progenitor cells.

[0070] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:16 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 2360 of SEQID NO:16, b is an integer of 15 to 2374, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:16, and whereb is greater than or equal to a +14.

[0071] Features of Protein Encoded by Gene NO: 7

[0072] The translation product of this gene shares sequence homologywith mitochondrial NADH-Ubiquinone oxidoreductase, chain 2.

[0073] Preferred polypeptides of the invention comprise the followingamino acid sequence: HFIITLTTFFTNYFL (SEQ ID NO: 271), and/orMKITFQDLFPMWNSFKCFLHGNVFSLFVLFPLLTCFSFPYTVNSGTKLDWVG WLVGWFFLEFMYINKGFEVTSENNISKRVLVRENIRIKSSPERVLRM (SEQ ID NO: 272).Polynucleotides encoding these polypeptides are also provided.

[0074] This gene is expressed primarily in stromal cells (cell codeTF274), induced epithelial cells and human cerebellum.

[0075] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, metabolicdisorders and conditions. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe liver, brain, and integument, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., cancerous and wounded tissues) orbodily fluids (e.g., lymph, bile, serum, plasma, urine, synovial fluidand spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0076] The tissue distribution in epithelial and cerebral tissuescombined with the homology to a known mitochondrial NADH-Ubiquinoneoxidoreductase gene indicates that polynucleotides and polypeptidescorresponding to this gene are useful for the diagnosis, prevention,and/or treatment of various metabolic disorders such as Tay-Sach'sdisease, phenylkenonuria, galactosemia, porphyrias, and Hurler'ssyndrome.

[0077] Additionally, the tissue distribution indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and diagnosis of hematopoietic related disorders suchas anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia, sincestromal cells are important in the production of cells of hematopoieticlineages. Furthermore, the protein may also be used to determinebiological activity, to raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0078] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:17 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1581 of SEQID NO:17, b is an integer of 15 to 1595, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:17, and whereb is greater than or equal to a +14.

[0079] Features of Protein Encoded by Gene NO: 8

[0080] The translation product of this gene shares sequence homologywith Platelet activating factor acetylhydrolase which inactivatesPlatelet activating factor, a potent phospholipid mediator affectingvarious physiological processes (See, e.g., Genbank Accession Nos.gi|349824|gb|AAA02880.1| and gi|2072303|gb|AAC04610.1|; all referencesavailable through this accession are hereby incorporated by referenceherein).

[0081] Preferred polypeptides of the invention comprise the followingamino acid sequence: RFWGSYEPHFSQEVSVIPP (SEQ ID NO: 273), and/orIRGNYFSGRKKSSSDTPKGSKDKISVWNRSQXACIRICKVHPNYIQIYLWHSAT SF (SEQ ID NO:274). Polynucleotides encoding these polypeptides are also provided.

[0082] This gene is expressed primarily in CD34 depleted buffy coat(cord blood) and to a lesser extent in human prostate cancer, stage 3fraction.

[0083] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, cancer,particularly of the prostate. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe immune system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., prostate, cancerous and wounded tissues) or bodily fluids (e.g.,lymph, cord blood, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0084] The tissue distribution in CD34 depleted buffy coat combined withthe homology to Platelet-activating factor acetylhydrolases, proteinsinvolved in regulation of platelet activity, indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Expression of this gene product in hematopoietic cells indicates a rolein the regulation of the proliferation; survival; differentiation;and/or activation of potentially all hematopoietic cell lineages,including blood stem cells. Representative uses are described in the“Immune Activity” and “Infectious Disease” sections below, in Example11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.

[0085] This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer e.g. by boosting immuneresponses. Expression in cells of lymphoid origin, the natural geneproduct may be involved in immune functions. Therefore it may be alsoused as an agent for immunological disorders including arthritis,asthma, immune deficiency diseases such as AIDS, and leukemia.Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0086] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:18 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1273 of SEQID NO:18, b is an integer of 15 to 1287, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:18, and whereb is greater than or equal to a +14.

[0087] Features of Protein Encoded by Gene NO: 9

[0088] Preferred polypeptides of the invention comprise the followingamino acid sequence:AGNQVEPFHVSLPSCLSPLPHLGHSMGVPSPTAWPSLASFHTQKKARIRQEEE SPPLPSPQELAFSALRVFFRV (SEQ ID NO: 275). Polynucleotides encoding these polypeptides arealso provided.

[0089] This gene is expressed primarily in primary dendritic cells.

[0090] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,immunosuppression and cancer. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe immune system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g.,lymph, serum, plasma, urine, synovial fluid and spinal fluid) or anothertissue or cell sample taken from an individual having such a disorder,relative to the standard gene expression level, i.e., the expressionlevel in healthy tissue or bodily fluid from an individual not havingthe disorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO:140 as residues: Arg-20 toLys-44, Arg-59 to Arg-68, Trp-74 to Lys-86, Thr-91 to Val-102.Polynucleotides encoding said polypeptides are also provided.

[0091] The tissue distribution in primary dendritic cells indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Expression of this gene product in dendritic cells indicates a role inthe regulation of the proliferation; survival; differentiation; and/oractivation of potentially all hematopoietic cell lineages, includingblood stem cells. Representative uses are described in the “ImmuneActivity” and “Infectious Disease” sections below, in Example 11, 13,14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the usesinclude bone marrow cell ex-vivo culture, bone marrow transplantation,bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.

[0092] This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer e.g. by boosting immuneresponses. Expression in cells of lymphoid origin, the natural geneproduct may be involved in immune functions. Therefore it may be alsoused as an agent for immunological disorders including arthritis,asthma, immune deficiency diseases such as AIDS, and leukemia.Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0093] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:19 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1382 of SEQID NO:19, b is an integer of 15 to 1396, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:19, and whereb is greater than or equal to a +14.

[0094] Features of Protein Encoded by Gene NO: 10

[0095] The translation product of this gene shares sequence homologywith peptide/histidine transporter from Rattus norvegicus and otherpeptide transporters which are thought to be important in transportingamino acids and peptides into cells (See, e.g., Genbank Accession No.gb|AAD24570.1|AF121080_(—)1; all references available through thisaccession are hereby incorporated by reference herein).

[0096] Preferred polypeptides of the invention comprise the followingamino acid sequence: FIQQNISFLLGYSIPVGCVGLAFFIFLFATPVFITKPP (SEQ ID NO:276). Polynucleotides encoding these polypeptides are also provided.

[0097] The gene encoding the disclosed cDNA is believed to reside onchromosome 11. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 11.

[0098] This gene is expressed primarily in macrophages and to a lesserextent in other immune cells including primary dendritic cells,neutrophils, resting T-cells, B cell lymphomas) and lung and fetal liverspleen.

[0099] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, cancerand disorders, particularly of the immune and hematopoietic systems.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the immunesystem, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g.,immune, hematopoietic, and/or other tissues) or bodily fluids (e.g.,lymph, serum, plasma, urine, synovial fluid and spinal fluid) or anothertissue or cell sample taken from an individual having such a disorder,relative to the standard gene expression level, i.e., the expressionlevel in healthy tissue or bodily fluid from an individual not havingthe disorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO:141 as residues: Arg-23 toGln-30, Asp-37 to Asp-50, Glu-230 to Met-235, Pro-271 to Arg-281,Arg-306 to Ser-316, Ser-318 to Gly-325. Polynucleotides encoding saidpolypeptides are also provided.

[0100] The tissue distribution in macrophages and other immune cellsindicates that polynucleotides and polypeptides corresponding to thisgene are useful for the diagnosis and treatment of cancer and otherproliferative disorders. This gene product may be involved in theregulation of cytokine production, antigen presentation, or otherprocesses suggests a usefulness in the treatment of cancer (e.g., byboosting immune responses). Alternatively expression within embryonictissue and other cellular sources marked by proliferating cellsindicates that this protein may play a role in the regulation orcellular division. Additionally, the expression in hematopoietic cellsand tissues indicates that this protein may play a role in theproliferation, differentiation, and/or survival of hematopoietic celllineages. Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. In such an event, this gene may beuseful in the treatment of lymphoproliferative disorders, and in themaintenance and differentiation of various hematopoietic lineages fromearly hematopoietic stem and committed progenitor cells. Similarly,embryonic development also involves decisions involving celldifferentiation and/or apoptosis in pattern formation. Thus this proteinmay also be involved in apoptosis or tissue differentiation and couldagain be useful in cancer therapy. Furthermore, the protein may also beused to determine biological activity, raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0101] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:20 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1263 of SEQID NO:20, b is an integer of 15 to 1277, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:20, and whereb is greater than or equal to a +14.

[0102] Features of Protein Encoded by Gene NO: 11

[0103] The translation product of this gene shares sequence homologywith procollagen-proline dioxygenase, an apparently secreted proteinwhich is thought to be important in the formation of 4-hydroxyproline incollagens (See, e.g., Genbank Accession No. pir|A33832|DACHA; allreferences available through this accession are hereby incorporated byreference herein). Furthermore, the translation product has an EF-handdomain (Prosite PS00018) which is a calcium binding domain as found incalmodulin, calpain, spectrin alpha chain, etc., (See, e.g. GeneSeqAccession No.R78523; all references available through this accession arehereby incorporated by reference herein).

[0104] Preferred polypeptides of the invention comprise the followingamino acid sequence:VSAHHPSGADEGVTAXQILPTEEYEEAMSTMQVSQLDLFRLLDQNRDGHLQLREVLAQTRLGNGWWMTPESIQEMYAAIKADPDGDGVLSLQEFSNMDLRDFHKYMRSHKAESSELVRNSHHTWLYQGEGAHHIMRAIRQRVLRLTRLSPEIVELSEPLQVVRYGEGGHYHAHVDSGPVYPETICSHTKLVANESVPFETSCRYMTVLFYLNNVTGGGETVFPVADNRTYDEMSLIQDDVDLRDTRRHCDKGNLRVKPQQGTAVFWYNYLPDGQGWVGDVDDYSLHGGCLVTRGTKWIANNWINVDPSRARQALFQQEMARLAREGGTDSQP EWALDRAXXDARVEL (SEQ ID NO: 277), AVFWYN (SEQID NO: 278), TVLFYLNNVTGGGETVFP (SEQ ID NO: 279),DLFRLLDQNRDGHLQLREVLAQTRLGNGWWMTPESIQEMYAAIKADPDGDG VLSLQEFS (SEQ ID NO:280), VSAHHPSGADEGVTAXQILPTEEYEEAMSTMQVSQLDL (SEQ ID NO: 281),FRLLDQNRDGHLQLREVLAQTRLGNGWWMTPESIQEMY (SEQ ID NO: 282),AAIKADPDGDGVLSLQEFSNMDLRDFHKYMRSHKAESS (SEQ ID NO: 283),ELVRNSHHTWLYQGEGAHHIMRAIRQRVLRLTRLSPEI (SEQ ID NO: 284),VELSEPLQVVRYGEGGHYHAHVDSGPVYPETICSHTKL (SEQ ID NO: 285),VANESVPFETSCRYMTVLFYLNNVTGGGETVFPVADNR (SEQ ID NO: 286),TYDEMSLIQDDVDLRDTRRHCDKGNLRVKPQQGTAVFW (SEQ ID NO: 287),YNYLPDGQGWVGDVDDYSLHGGCLVTRGTKWIANNWIN (SEQ ID NO: 288), and/orVDPSRARQALFQQEMARLAREGGTDSQPEWALDRAXXDARVEL (SEQ ID NO: 289).Polynucleotides encoding these polypeptides are also provided.

[0105] The gene encoding the disclosed cDNA is believed to reside onchromosome 3. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 3.

[0106] This gene is expressed primarily in human endometrial tumor andto a lesser extent in brain, as well as a variety of other normal andcancerous tissues.

[0107] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,endometrial cancer, in addition to other proliferative disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of thereproductive and neural systems, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., neural, reproductive, and/or othertissues) or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma,urine, synovial fluid and spinal fluid, lymph) or another tissue or cellsample taken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.Preferred polypeptides of the present invention comprise immunogenicepitopes shown in SEQ ID NO:142 as residues: Ser-21 to His-33, Ala-35 toThr-43. Polynucleotides encoding said polypeptides are also provided.

[0108] The tissue distribution in endometrial tumors combined with thehomology to procollagen-proline dioxygenase indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor diagnosis, treatment and prevention of these tumors, in addition toother tumors where expression has been indicated. The polypeptides ofthe invention is a good target for antagonists, particularly smallmolecules or antibodies, which block binding of the receptor by itscognate ligand(s). Accordingly, preferred are antibodies and or smallmolecules which specifically bind an extracellular portion of thetranslation product of this gene.

[0109] Also provided is a kit for detecting endometrial cancer. Such akit comprises in one embodiment an antibody specific for the translationproduct of this gene bound to a solid support. Also provided is a methodof detecting endometrial cancer in an individual which comprises a stepof contacting an antibody specific for the translation product of thisgene to a bodily fluid from the individual, preferably serum, andascertaining whether antibody binds to an antigen found in the bodilyfluid. Preferably the antibody is bound to a solid support and thebodily fluid is serum. Additionally, the homology to a conservedcollagen metabolizing protein would suggest that this protein may alsobe important in the diagnosis or treatment of various autoimmunedisorders such as rheumatoid arthritis, lupus, scleroderma, anddermatomyositis as well as dwarfism, spinal deformation, and specificjoint abnormalities as well as chondrodysplasias i.e. spondyloepiphysealdysplasia congenita, familial osteoarthritis, Atelosteogenesis type II,metaphyseal chondrodysplasia type Schmid. Furthermore, the protein mayalso be used to determine biological activity, raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0110] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:21 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1767 of SEQID NO:21, b is an integer of 15 to 1781, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:21, and whereb is greater than or equal to a +14.

[0111] Features of Protein Encoded by Gene NO: 12

[0112] This gene is expressed primarily in human osteoblastoma celllines (5/23 unique sequences) and to a lesser extent in T cells (4/23).

[0113] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,osteoblastoma, and other bone-related disorders. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the skeletal system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., bone and/or other tissues) orbodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0114] The tissue distribution in tumors of bone origins indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor diagnosis and intervention of these tumors, in addition to othertumors where expression has been indicated. Additionally, this gene is agood target for antagonists, particularly small molecules or antibodies,which block binding of the receptor by its cognate ligand(s).Accordingly, preferred are antibodies and or small molecules whichspecifically bind an extracellular portion of the translation product ofthis gene. The extracellular regions can be ascertained from theinformation regarding the transmembrane domains as set out above.

[0115] Also provided is a kit for detecting osteoblastoma and other bonerelated cancers. Such a kit comprises in one embodiment an antibodyspecific for the translation product of this gene bound to a solidsupport. Also provided is a method of detecting bone related cancers inan individual which comprises a step of contacting an antibody specificfor the translation product of this gene to a bodily fluid from theindividual, preferably serum, and ascertaining whether antibody binds toan antigen found in the bodily fluid. Preferably the antibody is boundto a solid support and the bodily fluid is serum. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0116] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:22 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1477 of SEQID NO:22, b is an integer of 15 to 1491, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:22, and whereb is greater than or equal to a +14.

[0117] Features of Protein Encoded by Gene NO: 13

[0118] The translation product of this gene is a human homolog of themouse acetylcholine receptor gamma chain, and is almost identical to ahuman acetylcholine receptor gamma chain (See, e.g., Genbank AccessionNos.: embICAA27442.11 and gb|AAA51568.1|; all references availablethrough these accessions are hereby incorporated by reference herein)which is thought to be important in transmission of nerve impulses tomuscles.

[0119] Preferred polypeptides of the invention comprise the followingamino acid sequence: LLADLMRNYDPHLRP (SEQ ID NO: 290),ISVTYFPFDWQNCSLIFQS (SEQ ID NO: 291), SMARGVRKVFLRLLPQ (SEQ ID NO: 292),QASPAIQACVDACNLMAR (SEQ ID NO: 293), and/or YNQVPDLPFPGDPRPYL (SEQ IDNO: 294). Polynucleotides encoding these polypeptides are also provided.This gene maps to chromosome 2, and therefore, may be used as a markerin linkage analysis for chromosome 2.

[0120] Included in this invention as preferred domains areNeurotransmitter-gated ion-channels domains, which were identified usingthe ProSite analysis tool. Structurally, members of the family ofNeurotransmitter-gated ion-channels are composed of a largeextracellular glycosylated N-terminal ligand-binding domain, followed bythree hydrophobic transmembrane regions which form the ionic channel,followed by an intracellular region of variable length. A fourthhydrophobic region is found at the C-terminal of the sequence. In theN-terminal extracellular domain of AchR/GABA/5HT3/Gly receptors, thereare two conserved cysteine residues, which, in AchR, have been shown toform a disulfide bond essential to the tertiary structure of thereceptor. A number of amino acids between the two disulfide-bondedcysteines are also conserved. We have therefore used this region as asignature pattern for this subclass of proteins. The consensus patternis as follows: C-x-[LIVMFQ]-x-[LIVMF]-x(2)-[FY]-P-x-D-x(3)-C.

[0121] Preferred polypeptides of the invention comprise the followingamino acid sequence: CSISVTYFPFDWQNC (SEQ ID NO: 295). Polynucleotidesencoding these polypeptides are also provided. Further preferred arepolypeptides comprising the Neurotransmitter-gated ion-channel domain ofthe amino acid sequence referenced in Table 1 for this gene, and atleast 5, 10, 15, 20, 25, 30, 50, or 75 additional contiguous amino acidresidues of the amino acid sequence referenced in Table 1 for this gene.The additional contiguous amino acid residues may be N-terminal orC-terminal to the Neurotransmitter-gated ion-channel domain.Alternatively, the additional contiguous amino acid residues may be bothN-terminal and C-terminal to the Neurotransmitter-gated ion-channeldomain, wherein the total N- and C-terminal contiguous amino acidresidues equal the specified number. The above preferred polypeptidedomain is characteristic of a signature specific toNeurotransmitter-gated ion-channels. Additionally, the polypeptide ofthis gene has been determined to have transmembrane domains at aboutamino acid positions 311-327, 248-264, 477-493, and 276-292. of theamino acid sequence referenced in Table 1 for this gene. Based uponthese characteristics, it is believed that the protein product of thisgene shares structural features to type Ma membrane proteins.

[0122] This gene is expressed primarily in fetal tissues (56/58 uniquesequences), specifically lung (42/58) and Dura Mater (14/58). It wasalso detected (1 sequence each) in a differentially expressed humancerebellum library and human tonsil library

[0123] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly fetal lung and brain, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues and cell types (e.g., developmental, neural,differentiating, and/or other tissues) or bodily fluids (e.g., lymph,serum, plasma, urine, synovial fluid and spinal fluid, pulmonarysurfactant) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder. Preferred polypeptides of thepresent invention comprise immunogenic epitopes shown in SEQ ID NO: 144as residues: Met-1 to Pro-7, Gln-21 to Glu-27, Arg-35 to Asp-49, Asn-66to Leu-72, Trp-82 to Glu-95, Pro-158 to Asn-163. Polynucleotidesencoding said polypeptides are also provided.

[0124] The tissue distribution in dura mater combined with the homologyto a conserved acetylcholine receptor indicates that polynucleotides andpolypeptides corresponding to this gene are useful for the detection,treatment, and/or prevention of neurodegenerative disease states,behavioral disorders, or inflammatory conditions. Representative usesare described in the “Regeneration” and “Hyperproliferative Disorders”sections below, in Example 11, 15, and 18, and elsewhere herein.Briefly, the uses include, but are not limited to the detection,treatment, and/or prevention of Alzheimer's Disease, Parkinson'sDisease, Huntington's Disease, schizophrenia, mania, dementia, paranoia,obsessive compulsive disorder, panic disorder, learning disabilities,ALS, psychoses, autism, and altered behaviors, including disorders infeeding, sleep patterns, balance, and perception. Potentially, this geneproduct is involved in synapse formation, neurotransmission, learning,cognition, homeostasis, or neuronal differentiation or survival. Inaddition, the gene or gene product may also play a role in the treatmentand/or detection of developmental disorders associated with thedeveloping embryo, and/or disorders of the cardiovascular and pulmonarysystems. Furthermore, the protein may also be used to determinebiological activity, to raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0125] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:23 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1825 of SEQID NO:23, b is an integer of 15 to 1839, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:23, and whereb is greater than or equal to a +14.

[0126] Features of Protein Encoded by Gene NO: 14

[0127] Preferred polypeptides of the invention comprise the followingamino acid sequence: VLKYALFLVLKNYYYCPY (SEQ ID NO: 296).Polynucleotides encoding these polypeptides are also provided. Thepolypeptide of this gene has been determined to have transmembranedomains at about amino acid positions 2945 and 8-24 of the amino acidsequence referenced in Table 1 for this gene. Based upon thesecharacteristics, it is believed that the protein product of this geneshares structural features to type IIIa membrane proteins.

[0128] This gene is expressed primarily in small intestine and to alesser extent in lung cancer.

[0129] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,gastrointestinal and pulmonary disorders. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the intestinal and pulmonary systems, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., gastrointestinal, pulmonary,and/or other tissues) or bodily fluids (e.g., serum, plasma, urine,synovial fluid and spinal fluid, lymph, and/or pulmonary surfactant) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder.

[0130] The tissue distribution in small intestine indicates a role inthe detection and/or treatment of gastrointestinal disorders includingWhipple's disease, Ulcers, and indigestion. Expression in the lungindicates a potential role in the treatment and/or detection of certainpulmonary defects such as pulmonary edema and embolism, bronchitis,cystic fibrosis and lung cancer. Furthermore, the protein may also beused to determine biological activity, to raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0131] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:24 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1370 of SEQID NO:24, b is an integer of 15 to 1384, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:24, and whereb is greater than or equal to a +14.

[0132] Features of Protein Encoded by Gene NO: 15

[0133] The translation product of this gene shares sequence homologywith mouse ECSIT (evolutionarily conserved signaling intermediate inToll pathways: see, e.g., Genbank accession number AF18210.1; allreferences available through this accession are hereby incorporated byreference herein.) which is thought to be important in innate immuneresponses. More specifically, ECSIT is believed to be novel intermediatein these signaling pathways that bridges TRAF6 to MEKK-1. This adapterprotein is specific for the Toll/IL-1 pathways and is a regulator ofMEKK-1 processing. Expression of wild-type ECSIT accelerates processingof MEKK-1, whereas a dominant-negative fragment of ECSIT blocks MEKK-1processing and activation of NF-kappaB. These results indicate animportant role for ECSIT in signaling to NF-kappaB and suggest thatprocessing of MEKK-1 is required for its function in the Toll/IL-1pathway (e.g., Kopp et al., Genes Dev 1999 Aug. 15;13(16):2059-71; thisreference is hereby incorporated by reference in its entirety herein.).Based on the sequence similarity, the translation product of this geneis expected to share at least some biological activities with ECSITproteins. Such activities are known in the art, some of which aredescribed elsewhere herein.

[0134] In another embodiment, polypeptides of the invention comprise thefollowing amino acid sequence:MREYGVERDLAVYNQLLNIFPKEVFRPRNIIQRIFVHYPRQQECGIAVLEQMENHGVMPNKETEFLLIQIFGRKSYPMLKLVRLKLWFPRFMNVNPFPVPRDLPQDPVELAMFGLRHMEPDLSARVTIYQVPLPKDSTGAADPPQPHIVGIQSPDQQAALARHNPARPVFVEGPFSLWLRNKCVYYHILRADLLPPEEREVEETPEEWNLYYPMQLDLEYVRSGWDNYEFDINEVEEGPVFAMCMAGAHDQATMAKWIQGLQETNPTLAQIPVVFRLAGSTRELQTSSAGLEEPPLPEDHQEEDDNLQRQQQG QS (SEQ ID NO:297). Polynucleotides encoding these polypeptides are also provided.

[0135] This gene is expressed primarily in brain and to a lesser extentin pancreas, testes, and other tissue types.

[0136] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,neurological, behavioral, gastrointestinal, and endocrine disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the nervoussystem, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g., brain,endocrine, and/or other tissues) or bodily fluids (e.g., serum, plasma,urine, synovial fluid and spinal fluid, and lymph) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 146 as residues: Val-33 toArg-39, Ser-57 to Thr-66, Pro-80 to Lys-86, Pro-155 to Cys-160, Val-215to Pro-223, Pro-250 to Gly-255, Pro-311 to Glu-323, Arg-338 to Tyr-344,Ser-396 to Gln-401, Pro-410 to Ser-431. Polynucleotides encoding saidpolypeptides are also provided.

[0137] The tissue distribution (e.g., fetal tissue) and homology toECSIT indicates that polynucleotides and polypeptides corresponding tothis gene are useful for innate immune responses and/or Toll/IL-1pathways and/or regulation of MEKK-1 processing.

[0138] The tissue distribution in brain indicates that polynucleotidesand polypeptides corresponding to this gene are useful for thedetection/treatment of neurodegenerative disease states and behavioraldisorders such as Alzheimer's Disease, Parkinson's Disease, Huntington'sDisease, schizophrenia, mania, dementia, paranoia, obsessive compulsivedisorder, panic disorder, learning disabilities, ALS, psychoses, autism,and altered behaviors, including disorders in feeding, sleep patterns,balance, and perception. In addition, the gene or gene product may alsoplay a role in the treatment and/or detection of developmental disordersassociated with the developing embryo, sexually-linked disorders, ordisorders of the cardiovascular system. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0139] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:25 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1667 of SEQID NO:25, b is an integer of 15 to 1681, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:25, and whereb is greater than or equal to a +14.

[0140] Features of Protein Encoded by Gene NO: 16

[0141] The translation product of this gene shares sequence homologywith the acid labile subunit of the insulin like growth factor bindingsubunit which is thought to be important in modulating the activity ofInsulin like growth factor. In addition, this gene also shares homologywith the melibiose carrier protein (thiomethylgalactoside permease II)of Caenorhabditis elegans (See Genbank Accession No. gi|1280135; allreferences available through this accession are hereby incorporated byreference herein).

[0142] Preferred polypeptides of the invention comprise the followingamino acid sequence: FQFGWASTQISHLSLIPEL (SEQ ID NO: 298),LRYAFTVVANITVY (SEQ ID NO: 299), FVYGSMSFLDKVANGLA (SEQ ID NO: 300),WHLVGTVCVLLSFPFIF (SEQ ID NO: 301), and/or GHFLNDLCASMWFTY (SEQ ID NO:302). Polynucleotides encoding these polypeptides are also provided.

[0143] This gene is expressed primarily in macrophages and to a lesserextent in dendritic cells.

[0144] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immuneand hematopoeitic disorders. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe hematopoetic and/or immune systems, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., hematopoeitic, immune, and/or othertissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluidand spinal fluid, and lymph) or another tissue or cell sample taken froman individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO:147 as residues: Ala-28 to Ala-33, Arg-38 to Leu-48, Thr-120 toLys-125, Gly-155 to Gln-163, Gly-200 to Glu-214. Polynucleotidesencoding said polypeptides are also provided.

[0145] The tissue distribution predominantly in dendritic cells andmacrophages combined with homology to a growth factor binding subunitindicates that polynucleotides and polypeptides corresponding to thisgene are useful for the treatment and diagnosis of hematopoietic relateddisorders such as anemia, pancytopenia, leukopenia, thrombocytopenia orleukemia since stromal cells are important in the production of cells ofhematopoietic lineages. Representative uses are described in the “ImmuneActivity” and “Infectious Disease” sections below, in Example 11, 13,14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the usesinclude bone marrow cell ex vivo culture, bone marrow transplantation,bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.

[0146] The gene product may also be involved in lymphopoiesis,therefore, it can be used in immune disorders such as infection,inflammation, allergy, immunodeficiency etc. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Furthermore,the protein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0147] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:26 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1935 of SEQID NO:26, b is an integer of 15 to 1949, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:26, and whereb is greater than or equal to a +14.

[0148] Features of Protein Encoded by Gene NO: 17

[0149] Preferred polypeptides of the invention comprise the followingamino acid sequence: AIPLRVLVVLWAFVLGLSRVMLGRHNVTDVAFGFFLGYMQ (SEQ IDNO: 303), and/or VGLSRVLGRHTDV (SEQ ID NO: 304). Polynucleotidesencoding these polypeptides are also provided.

[0150] The gene encoding the disclosed cDNA is believed to reside onchromosome 9. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 9.

[0151] This gene is expressed primarily in placenta and small intestine.

[0152] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,pregnancy, reproductive, and/or gastrointestinal disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the intestinal and endocrine systems,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types (e.g., reproductive,gastrointestinal, and/or other tissues) or bodily fluids (e.g., lymph,serum, plasma, urine, synovial fluid and spinal fluid, amniotic fluid,)or another tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder.

[0153] The tissue distribution in placenta indicates a potential rolefor this protein in the detection and/or treatment of pregnancydisorders such as miscarriage and/or gastro-intestinal disorders such asindigestion, ulcers and Whipple's disease. Alternatively,polynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis, prevention, and/or treatment of various metabolicdisorders such as Tay-Sachs disease, phenylkenonuria, galactosemia,porphyrias, and Hurler's syndrome. Furthermore, the protein may also beused to determine biological activity, to raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0154] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:27 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 2272 of SEQID NO:27, b is an integer of 15 to 2286, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:27, and whereb is greater than or equal to a +14.

[0155] Features of Protein Encoded by Gene NO: 18

[0156] Preferred polypeptides of the invention comprise the followingamino acid sequence: SFYKMKRNSYDRLRKVV (SEQ ID NO: 305). Polynucleotidesencoding these polypeptides are also provided.

[0157] The gene encoding the disclosed cDNA is believed to reside onchromosome 1. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 1. The polypeptideof this gene has been determined to have a transmembrane domain at aboutamino acid position 8-24 of the amino acid sequence referenced in Table1 for this gene. Moreover, a cytoplasmic tail encompassing amino acids25-51 of this protein has also been determined. Based upon thesecharacteristics, it is believed that the protein product of this geneshares structural features to type Ib membrane proteins.

[0158] This gene is expressed primarily in prostate and spleen and to alesser extent in most cell types.

[0159] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, prostateand immune disorders. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune and endocrine systems, expression of this gene at significantlyhigher or lower levels may be routinely detected in certain tissues orcell types (e.g., reproductive, immune, and/or other tissues) or bodilyfluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid,seminal fluid, and lymph) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0160] The tissue distribution in prostate indicates a potential role inthe treatment and/or detection of prostate disorders including benignprostate hyperplasia and prostate cancer. Expression in spleen indicatesa role in the treatment and/or detection of spleen disorders such assplenitis and spleen cancer.

[0161] Alternatively, the expression in the spleen may suggest thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Expression of this gene product intonsils indicates a role in the regulation of the proliferation;survival; differentiation; and/or activation of potentially allhematopoietic cell lineages, including blood stem cells. This geneproduct may be involved in the regulation of cytokine production,antigen presentation, or other processes that may also suggest ausefulness in the treatment of cancer e.g. by boosting immune responses.Expression in cells of lymphoid origin, the natural gene product may beinvolved in immune functions. Therefore it may be also used as an agentfor immunological disorders including arthritis, asthma, immunedeficiency diseases such as AIDS, and leukemia. Furthermore, the proteinmay also be used to determine biological activity, to raise antibodies,as tissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues. In addition, this gene product mayhave commercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types.

[0162] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:28 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 516 of SEQID NO:28, b is an integer of 15 to 530, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:28, and where bis greater than or equal to a +14.

[0163] Features of Protein Encoded by Gene No: 19

[0164] This gene was shown to have homology to both a human IgE-bindingprotein as well as to the human gene for Human Factor XIII (See GenbankAccession Nos. gb|S76337|S76337 and Q25893, respectively).

[0165] Preferred polypeptides of the invention comprise the followingamino acid sequence: LHQLRPPHRFPLIPPAAAEGAGAPPGCGYCVFWLLNPLP (SEQ ID NO:306), and/or MPWKRAVVLLMLWFIGQAMWLAPAYVLEFQGKNTFLFIWLAGLFFLLINCSILIQIISHYKEEPLTERIKYD (SEQ ID NO: 307). Polynucleotides encoding thesepolypeptides are also provided.

[0166] This gene is expressed primarily in infant brain and fetalcochlea.

[0167] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,neurological, behavioral, and hearing disorders as well as disorders ofthe somatosensory and auditory cortices. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the nervous system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., neural, immune, auditory, and/orother tissues) or bodily fluids (e.g., serum, plasma, urine, synovialfluid and spinal fluid, and lymph) or another tissue or cell sampletaken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.

[0168] The tissue distribution in infant brain indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor detection, treatment, and/or prevention of neurodegenerative diseasestates, behavioral disorders, or inflammatory conditions. Representativeuses are described in the “Regeneration” and “HyperproliferativeDisorders” sections below, in Example 11, 15, and 18, and elsewhereherein. Briefly, the uses include, but are not limited to the detection,treatment, and/or prevention of Alzheimer's Disease, Parkinson'sDisease, Huntington's Disease, schizophrenia, mania, dementia, paranoia,obsessive compulsive disorder, panic disorder, learning disabilities,ALS, psychoses, autism, and altered behaviors, including disorders infeeding, sleep patterns, balance, and perception. In addition, the geneor gene product may also play a role in the treatment and/or detectionof developmental disorders associated with the developing embryo,sexually-linked disorders, or disorders of the cardiovascular system.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tumors and tissues. Alternatively, considering the homology to aconserved human gene for IgE as well as to a conserved blood clottingfactor may suggest this gene is useful for the diagnosis and treatmentof a variety of immune system disorders. Homology of this gene to ablood clotting factor, specifically, indicates a role in the regulationof the proliferation; survival; differentiation; and/or activation ofpotentially all hematopoietic cell lineages, including blood stem cells.This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer e.g., by boosting immuneresponses. Since the gene may be expressed in cells of lymphoid origin,the natural gene product may be involved in immune functions. Thereforeit may be also used as an agent for immunological disorders includingarthritis, asthma, immune deficiency diseases such as AIDS, andleukemia. The tissue distribution in fetal cochlea indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor detection, treatment, and/or prevention of hearing disorders,cochlea dysfunction and/or disorders of the somatosensory and auditorycortices. Furthermore, the protein may also be used to determinebiological activity, to raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues. In addition, this gene product may have commercialutility in the expansion of stem cells and committed progenitors ofvarious blood lineages, and in the differentiation and/or proliferationof various cell types.

[0169] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:29 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1282 of SEQID NO:29, b is an integer of 15 to 1296, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:29, and whereb is greater than or equal to a +14.

[0170] Features of Protein Encoded by Gene No: 20

[0171] Preferred polypeptides of the invention comprise the followingamino acid sequence: ARAQPFAFQLRPAPGRPGSPVA (SEQ ID NO: 308),AGLPGALTAPAXHHHADSRPAELVVQPLSPPRPLLSHAGLASAAGASSLXRVPGEAESLCALSPGSALRFPAASCSRPXREPSGDEGTAGALPSPWLAALGPGGRPAVRRVLPRLGGRAGQLPRGLPVPRGLRHAGRYHLLRLLRAPLLLRRGRRQAGAGRLHQRPPRTGAPRHHCAACLRPLSHRRLHLHCVHHPGLCSGYLLLHLFETQGALAAANPLLTPQLSDRDPAHDPDLHQPQGTLPAVQHSHELQLHRRLHPQ VLLSHLVSWCHPSISLTPFSRSPHWLGRAVQTFSSX (SEQ ID NO: 309),AGLPGALTAPAXHHHADSRPAELVVQPLSPPRPLLSHA (SEQ ID NO: 310),GLASAAGASSLXRVPGEAESLCALSPGSALRFPAASCSRP (SEQ ID NO: 311),XREPSGDEGTAGALPSPWLAALGPGGRPAVRRVLPRLGGR (SEQ ID NO: 312),AGQLPRGLPVPRGLRHAGRYHLLRLLRAPLLLRRGRRQAG (SEQ ID NO: 313),AGRLHQRPPRTGAPRHHCAACLRPLSHRRLHLHCVHHPGL (SEQ ID NO: 314),CSGYLLLHLFETQGALAAANPLLTPQLSDRDPAHDPDLHQ (SEQ ID NO: 315), and/orPQGTLPAVQHSHELQLHRRLHPQVLLSHLVSWCHPSISLTPFSRSPHWLGRAV QTFSSX (SEQ ID NO:316). Polynucleotides encoding these polypeptides are also provided.

[0172] The gene encoding the disclosed cDNA is believed to reside onchromosome 4. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 4.

[0173] This gene is expressed primarily in heart and to a lesser extentin the embryo.

[0174] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,cardiovascular and developmental disorders. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the cardiovascular and developmental systems, expressionof this gene at significantly higher or lower levels may be routinelydetected in certain tissues or cell types (e.g., cardiopulmonary,developmental, and/or other tissues) or bodily fluids (e.g., lymph,sputum, serum, plasma, urine, synovial fluid and spinal fluid, amnioticfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder. Preferred polypeptides of thepresent invention comprise immunogenic epitopes shown in SEQ ID NO:151as residues: Gln-23 to Gly-30, Gln-35 to Gln-43, Leu-73 to Glu-84,Arg-125 to Pro-133, Ser-140 to Thr-145, Thr-153 to Thr-164.Polynucleotides encoding said polypeptides are also provided.

[0175] The tissue distribution in heart indicates that polynucleotidesand polypeptides corresponding to this gene are useful for the treatmentand/or detection of a range of vascular conditions, which include, butare not limited to, microvascular disease, vascular leak syndrome,aneurysm, stroke, atherosclerosis, arteriosclerosis, embolism,vasculitis, myocardial infarction, myocarditis, ischemia, stroke, inaddition to developmental and metabolic disorders. For example, thisgene product may represent a soluble factor produced by smooth musclethat regulates the innervation of organs or regulates the survival ofneighboring neurons. Likewise, it may be involved in controlling thedigestive process, and such actions as peristalsis. Similarly, it may beinvolved in controlling the vasculature in areas where smooth musclesurrounds the endothelium of blood vessels.

[0176] Alternatively, the expression in embryonic tissue indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of cancer and other proliferativedisorders. Furthermore, protein may play a role in the regulation ofcellular division. In such an event, this gene may be useful in thetreatment of lymphoproliferative disorders, and in the maintenance anddifferentiation of various hematopoietic lineages from earlyhematopoietic stem and committed progenitor cells. Similarly, embryonicdevelopment also involves decisions involving cell differentiationand/or apoptosis in pattern formation. Thus this protein may also beinvolved in apoptosis or tissue differentiation and could again beuseful in cancer therapy. Furthermore, the protein may also be used todetermine biological activity, raise antibodies, as tissue markers, toisolate cognate ligands or receptors, to identify agents that modulatetheir interactions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0177] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:30 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1965 of SEQID NO:30, b is an integer of 15 to 1979, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:30, and whereb is greater than or equal to a +14.

[0178] Features of Protein Encoded by Gene No: 21

[0179] The gene encoding the disclosed cDNA is believed to reside onchromosome 17. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 17.

[0180] This gene is expressed primarily in human teratocarcinoma cellline treated with retinoic acid, Hodgkin's Lymphoma human, and brain.

[0181] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,developmental abnormalities, neural disorders, or Hodgkin's disease.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the nervoussystem, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g.,developing, differentiating, neural, and/or other tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid, amniotic fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0182] The tissue distribution in teratocarcinoma cell line indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for early diagnosis and treatment of developmental abnormalities,including agenesis, aplasia, hypoplasia, dysraphic abnormalities,division failures, dysplasia, etc. Additionally, the gene and itsexpression can be used for teratogen detection or classification.

[0183] Alternatively, considering the expression within human braintissue may suggest that polynucleotides and polypeptides correspondingto this gene are useful for the detection/treatment of neurodegenerativedisease states and behavioral disorders such as Alzheimer's Disease,Parkinson's Disease, Huntington's Disease, schizophrenia, mania,dementia, paranoia, obsessive compulsive disorder, panic disorder,learning disabilities, ALS, psychoses, autism, and altered behaviors,including disorders in feeding, sleep patterns, balance, and perception.In addition, the gene or gene product may also play a role in thetreatment and/or detection of developmental disorders associated withthe developing embryo. Furthermore, the protein may also be used todetermine biological activity, to raise antibodies, as tissue markers,to isolate cognate ligands or receptors, to identify agents thatmodulate their interactions, in addition to its use as a nutritionalsupplement. Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tissues.

[0184] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:31 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1260 of SEQID NO:31, b is an integer of 15 to 1274, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:31, and whereb is greater than or equal to a +14.

[0185] Features of Protein Encoded by Gene No: 22

[0186] The translation product of this gene was shown to have homologyto the human B-cell growth factor which is known to be involved in thematuration of B-cells (See Genbank Accession No. gi|522145; allreferences available through this accession are hereby incorporated byreference herein).

[0187] Preferred polypeptides of the invention comprise the followingamino acid sequence: VAHTCNLSTLGGQGGRIERTAGQEFKTS (SEQ ID NO: 317) andHYKSYACRYRSGIRGRVDEVLTNCHWTYLKQNRKMAANSSGQALHSRDPLLIRTSGITLSSSILQPNRRQLCSMLMHIHLDTSSLKTLHLGTLFFLFYLALTQNEE NICDGKVTL (SEQID NO: 318). Also preferred are the polynucleotides encoding thesepolypeptides.

[0188] The gene encoding the disclosed cDNA is believed to reside onchromosome 9. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 9.

[0189] This gene is expressed primarily in multiple sclerosis andprostate tissues and to a lesser extent in brain and osteoblasts.

[0190] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, muscle,reproductive, bone and neural disorders. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the central nervous system and/or PNS, expression ofthis gene at significantly higher or lower levels may be routinelydetected in certain tissues or cell types (e.g., muscle, reproductive,and/or other tissues) or bodily fluids (e.g., lymph, serum, plasma,urine, synovial fluid and spinal fluid, seminal fluid) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO:153 as residues: Gln-28 toAsp-35. Polynucleotides encoding said polypeptides are also provided.

[0191] The tissue distribution in multiple sclerosis indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the detection, treatment, and/or prevention of neurodegenerativedisease states, behavioral disorders, or inflammatory conditions.Representative uses are described in the “Regeneration” and“Hyperproliferative Disorders” sections below, in Example 11, 15, and18, and elsewhere herein. Briefly, the uses include, but are not limitedto the detection, treatment, and/or prevention Alzheimer's Disease,Parkinson's Disease, Huntington's Disease, schizophrenia, mania,dementia, paranoia, obsessive compulsive disorder, panic disorder,learning disabilities, ALS, psychoses, autism, and altered behaviors,including disorders in feeding, sleep patterns, balance, and perception.In addition, the gene or gene product may also play a role in thetreatment and/or detection of developmental disorders associated withthe developing embryo, sexually-linked disorders, or disorders of thecardiovascular system. Furthermore, the protein may also be used todetermine biological activity, to raise antibodies, as tissue markers,to isolate cognate ligands or receptors, to identify agents thatmodulate their interactions, in addition to its use as a nutritionalsupplement. Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tissues.

[0192] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:32 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1517 of SEQID NO:32, b is an integer of 15 to 1531, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:32, and whereb is greater than or equal to a +14.

[0193] Features of Protein Encoded by Gene No: 23

[0194] The translation product of this gene was shown to have homologyto the B0035.14 gene of Caenorhabditis elegans (See, e.g., GenbankAccession No. gnl|PID|e242592; all references available through thisaccession are hereby incorporated by reference herein).

[0195] Preferred polypeptides of the invention comprise the followingamino acid sequence: TIKMQTENLGVVYYVNKDF (SEQ ID NO: 319), MVSNPPY (SEQID NO: 321), HASEL (SEQ ID NO: 322),RESWYACRYRSGIPGSTHASELMPIIVLILVSLLSQLMVSNPPYSLYPRSGTGQTIKMQTENLGVVYYVNKDFKNEYKGMLLQKVEKSVEEDYVTNIRNNCWKERQQKTDMQYAAKVYRDDRLRRRQMP (SEQ ID NO: 323) and/or VEEDYVTNIRNNC (SEQ IDNO: 320). Polynucleotides encoding these polypeptides are also provided.

[0196] The gene encoding the disclosed cDNA is believed to reside onchromosome 4. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 4.

[0197] This gene is expressed primarily in bone marrow and to a lesserextent in lung and various tissues.

[0198] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,hematopoietic, and/or cardiopulmonary disorders. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the hematopoietic system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., proliferating, haematopoeitic,and/or other tissues) or bodily fluids (e.g., lymph, serum, plasma,urine, synovial fluid and spinal fluid, pulmonary surfactant) or anothertissue or cell sample taken from an individual having such a disorder,relative to the standard gene expression level, i.e., the expressionlevel in healthy tissue or bodily fluid from an individual not havingthe disorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO:154 as residues: Ile-34 toGlu-39, Lys-49 to Lys-56, Val-63 to Glu-68, Thr-73 to Asp-88, Arg-97 toPro-107. Polynucleotides encoding said polypeptides are also provided.

[0199] The tissue distribution in bone marrow indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and diagnosis of hematopoietic related disorders suchas anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia sincestromal cells are important in the production of cells of hematopoieticlineages. Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Briefly, the uses include bone marrowcell ex vivo culture, bone marrow transplantation, bone marrowreconstitution, radiotherapy or chemotherapy of neoplasia.

[0200] The gene product may also be involved in lymphopoiesis,therefore, it can be used in immune disorders such as infection,inflammation, allergy, immunodeficiency, etc. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Furthermore,the protein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0201] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:33 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 2076 of SEQID NO:33, b is an integer of 15 to 2090, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:33, and whereb is greater than or equal to a +14.

[0202] Features of Protein Encoded by Gene No: 24

[0203] Preferred polypeptides of the invention comprise the followingamino acid sequence: LVALDRMEYVRTFRKREDLRGRLFWVALDLLDLLD (SEQ ID NO:324). Polynucleotides encoding these polypeptides are also provided. Thepolypeptide of this gene has been determined to have transmembranedomains at about amino acid positions 20-36 and 53-69 of the amino acidsequence referenced in Table 1 for this gene. Based upon thesecharacteristics, it is believed that the protein product of this geneshares structural features to type IIIa membrane proteins.

[0204] This gene is expressed primarily in T-cells and breast cancertissue.

[0205] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immunedisorders and breast cancer. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe immune system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., immune, breast, proliferating, and/or other tissues) or bodilyfluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid,breast milk, and lymph) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO: 155 as residues: Tyr-105 to Pro-113, Gln-122 to Pro-133, Pro-140 toAsp-155. Polynucleotides encoding said polypeptides are also provided.

[0206] The tissue distribution in T cells and breast cancer indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for the diagnosis and treatment of a variety of immune systemdisorders and breast cancer. Representative uses are described in the“Immune Activity” and “Infectious Disease” sections below, in Example11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, theexpression of this gene product in T-cells indicates a role in theregulation of the proliferation; survival; differentiation; and/oractivation of potentially all hematopoietic cell lineages, includingblood stem cells. This gene product may be involved in the regulation ofcytokine production, antigen presentation, or other processes that mayalso suggest a usefulness in the treatment of cancer e.g., by boostingimmune responses. Expression in cells of lymphoid origin, the naturalgene product may be involved in immune functions. Therefore it may bealso used as an agent for immunological disorders including arthritis,asthma, immune deficiency diseases such as AIDS, and leukemia.Furthermore, the protein may also be used to determine biologicalactivity, raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues. In addition, this gene product may have commercialutility in the expansion of stem cells and committed progenitors ofvarious blood lineages, and in the differentiation and/or proliferationof various cell types. The expression of the gene in the breast cancertissue may indicate T-cell mediated immune reaction to the cancertissue.

[0207] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:34 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 992 of SEQID NO:34, b is an integer of 15 to 1006, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:34, and whereb is greater than or equal to a +14.

[0208] Features of Protein Encoded by Gene No: 25

[0209] The translation product of this gene shares sequence homologywith an yeast ankyrin repeat-containing protein Akrlp which is thoughtto be important in pheromone response pathway (See Genbank Accession No.gi|466522; all references available through this accession are herebyincorporated by reference herein).

[0210] Preferred polypeptides of the invention comprise the followingamino acid sequence:SVALFYNFGKSWKSDPGIIKXTEEQKKKTIVELAETGSLDLSIFCSTCLIRKPVR SKHCGVCNRCIAKFDHHCPWVGNCVGAGNHRYF (SEQ ID NO: 325), FDHHCPWVGNCV (SEQ ID NO: 326),and/or QMYQISCLGITTNERMNARR (SEQ ID NO: 327). Polynucleotides encodingthese polypeptides are also provided.

[0211] The gene encoding the disclosed cDNA is believed to reside onchromosome 12. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 12. Thepolypeptide of this gene has been determined to have a transmembranedomain at about amino acid position 53-69 of the amino acid sequencereferenced in Table 1 for this gene. Moreover, a cytoplasmic tailencompassing amino acids 70-150 of this protein has also beendetermined. Based upon these characteristics, it is believed that theprotein product of this gene shares structural features to type Iamembrane proteins.

[0212] This gene is expressed primarily in human lung cancer cells,B-cell lymphoma and to a lesser extent in fetal tissues and tumor cellsof various origins.

[0213] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, cancer ofvarious origins, particularly of the lungs and hematopoietic systems.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the lung,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types (e.g., lung,cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma,urine, synovial fluid and spinal fluid, pulmonary surfactant, and lymph)or another tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder. Preferred polypeptides of the present inventioncomprise immunogenic epitopes shown in SEQ ID NO: 156 as residues:Thr-28 to Phe-35, Asp-140 to Ser-145. Polynucleotides encoding saidpolypeptides are also provided.

[0214] The tissue distribution in lung cancer indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Briefly, the expression of this geneproduct in lymphomas indicates a role in the regulation of theproliferation; survival; differentiation; and/or activation ofpotentially all hematopoietic cell lineages, including blood stem cells.This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer e.g., by boosting immuneresponses. Expression in cells of lymphoid origin, the natural geneproduct may be involved in immune functions. Therefore it may be alsoused as an agent for immunological disorders including arthritis,asthma, immune deficiency diseases such as AIDS, and leukemia. Protein,as well as, antibodies directed against the protein may show utility asa tumor marker and/or immunotherapy targets for the above listed tumorsand tissues. In addition, this gene product may have commercial utilityin the expansion of stem cells and committed progenitors of variousblood lineages, and in the differentiation and/or proliferation ofvarious cell types. Alternatively, distribution in tumor tissuesindicates that polynucleotides and polypeptides corresponding to thisgene are useful for diagnosis and treatment of cancers of variousorigins, especially lung B-cell lymphoma, stomach cancer, osteoclastoma.Additionally, this gene is a good target for antagonists, particularlysmall molecules or antibodies, which block binding of the receptor byits cognate ligand(s). Accordingly, preferred are antibodies and orsmall molecules which specifically bind an extracellular portion of thetranslation product of this gene. Also provided is a kit for detectinglung cancer. Such a kit comprises in one embodiment an antibody specificfor the translation product of this gene bound to a solid support. Alsoprovided is a method of detecting lung cancer in an individual whichcomprises a step of contacting an antibody specific for the translationproduct of this gene to a bodily fluid from the individual, preferablyserum, and ascertaining whether antibody binds to an antigen found inthe bodily fluid. Preferably the antibody is bound to a solid supportand the bodily fluid is serum. Furthermore, the protein may also be usedto determine biological activity, raise antibodies, as tissue markers,to isolate cognate ligands or receptors, to identify agents thatmodulate their interactions, in addition to its use as a nutritionalsupplement. Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tissues.

[0215] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:35 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1773 of SEQID NO:35, b is an integer of 15 to 1787, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:35, and whereb is greater than or equal to a +14.

[0216] Features of Protein Encoded by Gene No: 26

[0217] The gene encoding the disclosed cDNA is believed to reside onchromosome 15. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 15.

[0218] This gene is expressed primarily in infant brain, fetal tissue(e.g., heart), tumors and to a lesser extent in a variety of othertissues and cell types.

[0219] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,developmental and neurodegenerative diseases of the brain and nervoussystem, as well as, cancer, in general. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the brain, CNS, and/or PNS, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., developmental, differentiating,neural, and/or other tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 157 as residues: Ser-33 toIle-41. Polynucleotides encoding said polypeptides are also provided.

[0220] The tissue distribution in infant brain indicates polynucleotidesand polypeptides corresponding to this gene are useful for thedetection, treatment, and/or prevention of neurodegenerative diseasestates, behavioral disorders, or inflammatory conditions. Representativeuses are described in the “Regeneration” and “HyperproliferativeDisorders” sections below, in Example 11, 15, and 18, and elsewhereherein. Briefly, the uses include, but are not limited to the detection,treatment, and/or prevention of Alzheimer's Disease, Parkinson'sDisease, Huntington's Disease, Tourette's Syndrome, meningitis,encephalitis, demyelinating diseases, peripheral neuropathies,neoplasia, trauma, congenital malformations, spinal cord injuries,ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania,dementia, paranoia, obsessive compulsive disorder, depression, panicdisorder, learning disabilities, ALS, psychoses, autism, and alteredbehaviors, including disorders in feeding, sleep patterns, balance, andperception. In addition, elevated expression of this gene product inregions of the brain indicates it plays a role in normal neuralfunction. Potentially, this gene product is involved in synapseformation, neurotransmission, learning, cognition, homeostasis, orneuronal differentiation or survival.

[0221] Moreover, the expression within fetal, embryonic tissue and othercellular sources marked by proliferating cells indicates this proteinmay play a role in the regulation of cellular division, and may showutility in the diagnosis, treatment, and/or prevention of developmentaldiseases and disorders, including cancer, and other proliferativeconditions. Representative uses are described in the “HyperproliferativeDisorders” and “Regeneration” sections below and elsewhere herein.Briefly, developmental tissues rely on decisions involving celldifferentiation and/or apoptosis in pattern formation. Dysregulation ofapoptosis can result in inappropriate suppression of cell death, asoccurs in the development of some cancers, or in failure to control theextent of cell death, as is believed to occur in acquiredimmunodeficiency and certain degenerative disorders, such as spinalmuscular atrophy (SMA). Alternatively, this gene product may be involvedin the pattern of cellular proliferation that accompanies earlyembryogenesis. Thus, aberrant expression of this gene product intissues—particularly adult tissues—may correlate with patterns ofabnormal cellular proliferation, such as found in various cancers.Because of potential roles in proliferation and differentiation, thisgene product may have applications in the adult for tissue regenerationand the treatment of cancers. It may also act as a morphogen to controlcell and tissue type specification. Therefore, the polynucleotides andpolypeptides of the present invention are useful in treating, detecting,and/or preventing said disorders and conditions, in addition to othertypes of degenerative conditions. Thus this protein may modulateapoptosis or tissue differentiation and is useful in the detection,treatment, and/or prevention of degenerative or proliferative conditionsand diseases. The protein is useful in modulating the immune response toaberrant polypeptides, as may exist in proliferating and cancerous cellsand tissues. The protein can also be used to gain new insight into theregulation of cellular growth and proliferation. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0222] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:36 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1187 of SEQID NO:36, b is an integer of 15 to 1201, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:36, and whereb is greater than or equal to a +14.

[0223] Features of Protein Encoded by Gene No: 27

[0224] The translation product of this gene shares sequence homologywith a zinc transporter, ZnT-1, which is thought to regulate zincexcretion from cells and maintain homeostasis (See Genbank Accession No.gb|AAA79234.|, all references available through this accession arehereby incorporated by reference herein; as well as Palmiter andFindley, EMBO J. 14:639-649 (1995), which is hereby incorporated byreference herein). Transformation of normal cells with a mutant ratZnT-1 lacking the first membrane-spanning domain conferred zincsensitivity on wild-type cells, suggests that ZnT-1 functions as amultimer. Deletion of the first two membrane-spanning domains resultedin a non-functional molecule, whereas deletion of the C-terminal tailproduced a toxic phenotype. Transmembrane domains of the protein of thecurrent invention are predicted using PSORT to comprise the followingamino acid residues as shown in SEQ ID NO: : Ser-42 to Ala-58, Ala-83 toLeu-99, Leu-115 to Gly-131, Val-249 to Val-265, and/or Val-314 toLeu-330. Therefore, preferred polypeptides of the present invention arethe predicted extracellular domains, comprising the following amino acidsequence: RVTSSLAMLSDS (SEQ ID NO: 328), AIERFIEPHEMQQPL (SEQ ID NO:329), AGIRHERNRGRLLCMLALTFMFMVLEVVVSRVTSSLAMLSDSFHMLSDVLALVVALVAERFARRTHATQKNTFGWIRAEVMGALVNAIFLTGLCFAILLEAIERFEEPHEMQQPLVVLGVGVAGLLVNVLGLCLFHHHSGFSQDSGHXHSHGGHGHGHGLPKGPRVKSTRPGSSDINVAPGEQGPDQEETNTLVANTSNSNGLKLDPADPENPRSGDTVEVQVNGNLVREPDHMELEEDRAGQLNMRGVFLHVLGDALGSVIVVVNALVFYFSWKGCSEGDFCVNPCFPDPCKPFVEIINSTHASVYEAGPCWVLYLDPTLCVVMVCILLYTTYPLLKESALILLQTVPKQIDIRNLIKELRNVEGVEEVHELHVWQLAGSRIIATAHIKCEDPTSYMEVAKXIKDVFHNHGIHATTIQPEFASVGSKSSVVPCELACRTQCALKQCCGTLPQAPSGKDAEKTPAVSISCLELSNNLEKKPRRTKAENIP AVVIEIKNMPKQTT (SEQ ID NO: 332) and/orNALVFYFSWKGCSEGDFCVNPCFPDPCKPFVEIINSTHASVYEAGPCWV (SEQ ID NO: 330). Anadditional preferred polypeptide fragment of the invention comprises thefollowing amino acid sequence:AGIRHERNRGRLLCMLALTFMFMVLEVVVSRVTSSLAMLSDSFHMLSDVLALVVALVAERFARRTHATQKNTFGWIRAEVMGALVNAFLTGLCFAILLEAIERFIEPHEMQQPLVVLGVGVAGLLVNVLGLCLFHHHSGFSQDSGHXHSHGGHGHGHGLPKGPRVKSTRPGSSDINVAPGEQGPDQEETNTLVANTSNSNGLKLDPADPENPRSGDTVEVQVNGNLVREPDHMELEEDRAGQLNMRGVFLHVLGDALGSVIVVVNALVFYFSWKGCSEGDFCVNPCFPDPCKAFVEILIVLMHQFM (SEQ ID NO: 331).Polynucleotides encoding this sequence are also provided. Thepolypeptide of this gene has been determined to have multipletransmembrane domains (e.g., about amino acid position 69-85, 101-117,300-316, 235-251, and 28-44 of the amino acid sequence referenced inTable 1 for this gene). Based upon these characteristics, it is believedthat the protein product of this gene shares structural features to typeIIIa membrane proteins.

[0225] This gene is expressed primarily in colon, lung, liver, lymphoma,osteosarcoma, adrenal gland and parathyroid tumor and fibroblasts.

[0226] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, Hodgkin'sLymphoma, osteosarcoma, neurodegenerative disorders, gastrointestinaldisorders, and cancer of many tissues. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For anumber of disorders of the above tissues or cells,particularly of the central nervous system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., neural, gastrointestinal, and/orother tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO: 158 as residues: Arg-50 to Thr-58, Ser-125 to Gly-132.Polynucleotides encoding said polypeptides are also provided.

[0227] The tissue distribution and homology to ZnT-1 indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor treatment and diagnosis of disorders associated with the regulationof zinc homeostasis. Although zinc is an important trace element in manybiological systems, several lines of evidence suggest that thistransporter may serve as a point of intervention particularly in thetreatment of neurological diseases. The metabolism of zinc in the brainhas been shown to be regulated by a number of transport proteins,including ZnT-1. Pharmacological doses of zinc cause neuronal death, andsome estimates indicate that extracellular concentrations of zinc couldreach neurotoxic levels under pathological conditions. In Alzheimer'sdisease, zinc has been shown to aggregate beta-amyloid, a form which ispotentially neurotoxic. The zinc-dependent transcription factorsNF-kappa B and Spl bind to the promoter region of the amyloid precursorprotein (APP) gene. Zinc also inhibits enzymes which degrade APP tononamyloidogenic peptides and which degrade the soluble form ofbeta-amyloid. The changes in zinc metabolism which occur duringoxidative stress may be important in neurological diseases whereoxidative stress is implicated, such as Alzheimer's disease, Parkinson'sdisease, and amyotrophic lateral sclerosis (ALS). Zinc is a structuralcomponent of superoxide dismutase 1, mutations of which give rise to oneform of familiar ALS. After HIV infection, zinc deficiency is foundwhich may be secondary to immune-induced cytokine synthesis. Zinc isinvolved in the replication of the HIV virus at a number of sites.Collectively, this transporter may prove useful in the treatment anddiagnosis of several disorders related to zinc regulation.

[0228] Alternatively, the tissue distribution within lymphomas indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for the diagnosis and treatment of a variety of immune systemdisorders. Expression of this gene product in immune tissue indicates arole in the regulation of the proliferation; survival; differentiation;and/or activation of potentially all hematopoietic cell lineages,including blood stem cells. This gene product may be involved in theregulation of cytokine production, antigen presentation, or otherprocesses that may also suggest a usefulness in the treatment of cancere.g. by boosting immune responses. Expression in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,and leukemia. Furthermore, the protein may also be used to determinebiological activity, to raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues. In addition, this gene product may have commercialutility in the expansion of stem cells and committed progenitors ofvarious blood lineages, and in the differentiation and/or proliferationof various cell types.

[0229] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:37 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1882 of SEQID NO:37, b is an integer of 15 to 1896, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:37, and whereb is greater than or equal to a +14.

[0230] Features of Protein Encoded by Gene No: 28

[0231] The translation product of this gene was shown to have homologyto the mouse interferon-stimulated gene 15 and human calnexin (SeeGenbank Accession Nos. gb|AAB02697.1| and gi|30648|gb|AAA21013.1|; allreferences available through these accessions are hereby incorporated byreference herein) which may implicate this gene as playing a role inregulation of proliferating and differentiating cells. Preferredpolypeptides comprise the following amino acid sequence:MFTFASMTKEDSKLIALIWPSEWQMIQKLFVVDHVIKITRIEVGDVNPSETQYISEPKLCPECREGLLCQQQRDLREYTQATIYVHKVVDNKKVMKDSAPELNVSSSETEEDKEEAKPDGEKDPDFNQSXGGTKRQKISHQNYIAYQKQVIRRSMRHRKVRGEKALLVSANQTLKELKIQIMHAFSVAPFDQNLSIDGKILSDDCATLGTLGVIPESVILLKADEPIADYAAMDDVMQVCMPEEGFKGTGLLGH (SEQ ID NO: 333),SAPELNVSSSETEEDKEEAKP (SEQ ID NO: 334),FQDKNRPCLSNWPEDTDVLYIVSQFFVEEWRKFVRKPTRCSPVSSVGNSALLC PHGGLMFFASMTKEDSKLIALIWPSEWQMIQKLFVVDHVIKITRIEVGDVNPSETQYISEPKLCPECREGLLCQQQRDLREYTQATIYVHKVVDNKKVMKDSAPELNVSSSETEEDKEEAKPDGEKDPDFNQSXGGTKRQKISHQNYIAYQKQVIRRSMRHRKVRGEKALLVSANQTLKELKIQIMHAFSVAPFDQNLSIDGKILSDDCA TLGTLGVIPESVILLKADEPIADYAAMDDVMQVCMPEEGFKGTGLLGH (SEQ ID NO: 343),FQDKNRPCLSNWPEDTDVLYIVSQFFVEEWRKFVRKPTRCSPVSSVGNSALLC PHGGL (SEQ ID NO:336), MFTFASMTKEDSKLIALIWPSEWQMIQKLFVVDHVIKITRIEE (SEQ ID NO: 337),VGDVNPSETQYISEPKLCPECREGLLCQQQRDLREYTQATIY (SEQ ID NO: 338),VHKVVDNKKVMKDSAPELNVSSSETEEDKEEAKPDGEKDPDF (SEQ ID NO: 339),NQSXGGTKRQKISHQNYIAYQKQVIRRSMRHRKVRGEKALLV (SEQ ID NO: 340),SANQTLKELKIQIMHAFSVAPFDQNLSIDGKILSDDCATLGT (SEQ ID NO: 341),LGVIPESVILLKADEPIADYAAMDDVMQVCMPEEGFKGTGLLGH (SEQ ID NO: 342), and/orKELKIQIMHAFSVAPFDQ (SEQ ID NO: 335). Also preferred are thepolynucleotides encoding these polypeptides.

[0232] The gene encoding the disclosed cDNA is believed to reside onchromosome 1. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 1.

[0233] This gene is expressed primarily in brain, lung cancer, bonemarrow, tonsils and hematological tissues.

[0234] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, cancers,developmental and regulatory diseases of the brain and immune system.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the brain andimmune system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO:159 as residues: His-26 toPhe-31. Polynucleotides encoding said polypeptides are also provided.

[0235] The tissue distribution in brain indicates that polynucleotidesand polypeptides corresponding to this gene are useful for thedetection, treatment, and/or prevention of neurodegenerative diseasestates, behavioral disorders, or inflammatory conditions. Representativeuses are described in the “Regeneration” and “HyperproliferativeDisorders” sections below, in Example 11, 15, and 18, and elsewhereherein. Briefly, the uses include, but are not limited to the detection,treatment, and/or prevention of Alzheimer's Disease, Parkinson'sDisease, Huntington's Disease, Tourette Syndrome, meningitis,encephalitis, demyelinating diseases, peripheral neuropathies,neoplasia, trauma, congenital malformations, spinal cord injuries,ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania,dementia, paranoia, obsessive compulsive disorder, depression, panicdisorder, learning disabilities, ALS, psychoses, autism, and alteredbehaviors, including disorders in feeding, sleep patterns, balance, andperception. In addition, expression in T-cells and bone marrow, andhomology to the mouse interferon-stimulated gene 15 and human calnexinproteins indicate that the protein product of this gene might also beuseful for the diagnosis and treatment of immune disorders including:leukemias, lymphomas, auto-immunities, immunodeficiencies (e.g., AIDS),immuno-supressive conditions (transplantation) and hematopoeiticdisorders. This gene product may be involved in the regulation ofcytokine production, antigen presentation, or other processes suggests ausefulness in the treatment of general microbial infection,inflammation, and cancer (e.g., by boosting immune responses). Thetissue distribution in bone marow, tonsils, and other immune tissues,indicates polynucleotides and polypeptides corresponding to this geneare useful for the diagnosis and treatment of a variety of immune systemdisorders. Representative uses are described in the “Immune Activity”and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18,19, 20, and 27, and elsewhere herein. Briefly, the expression indicatesa role in regulating the proliferation; survival; differentiation;and/or activation of hematopoietic cell lineages, including blood stemcells. Involvement in the regulation of cytokine production, antigenpresentation, or other processes indicates a usefulness for treatment ofcancer (e.g. by boosting immune responses). Expression in cells oflymphoid origin, indicates the natural gene product would be involved inimmune functions. Therefore it would also be useful as an agent forimmunological disorders including arthritis, asthma, immunodeficiencydiseases such as AIDS, leukemia, rheumatoid arthritis, granulomatousdisease, inflammatory bowel disease, sepsis, acne, neutropenia,neutrophilia, psoriasis, hypersensitivities, such as T-cell mediatedcytotoxicity; immune reactions to transplanted organs and tissues, suchas host-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.

[0236] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. Thus, this gene productis thought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0237] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:38 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1138 of SEQID NO:38, b is an integer of 15 to 1152, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:38, and whereb is greater than or equal to a +14.

[0238] Features of Protein Encoded by Gene No: 29

[0239] Preferred polypeptides of the invention comprise the followingamino acid sequence: RGERSEELLGREGLSGSQ (SEQ ID NO: 344), 1GGQDGHFTSTCVLALPRHACHFWGSLGVTVTRRAVQPRKSTLALHSPNPSALQTQCSSILCCHSTLGHAMQMQLEQAPVYCSXRSPQRCILPHGNMGSTCPGNRWEGRGSCCPQAPATAASASVAGMVAVGVVVVVXVVRXVAGVVVVVEAHIRHMRYVARMTVMVKDSQVAPPPEGPRLGPADSVSPCSCTVPLHVTVLPSVEKAGGQQQQQQQDRHSSTCDPSHEGCAPQEAQHLGAGQSLSAQQLLTPFSPSA ASAQPSQSLNFV (SEQID NO: 346), and/or AEAAEGEKGVRSCWAERDCPAPRCWASWGAQPSWDGSQVLLWRSCCCCCCWPPAFSTDGRTVTWRGTVQLQGETESAGPSLGPSGGGATWESFTITVILATYLMCRMWASTTTTTPATXLTTTTTT PTATIPATLAEAAVAGACGQQLPLPSHLFPGQVDPMFPCGRMHLWGERXEQ (SEQ ID NO: 345). Polynucleotides encodingthese polypeptides are also provided.

[0240] This gene is expressed primarily in placenta, salivary gland, andcolon.

[0241] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,developmental anomalies or fetal deficiencies and/or disorders of thecolon. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thedeveloping fetus, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., reproductive, and/or other tissues) or bodily fluids (e.g.,lymph, serum, plasma, urine, synovial fluid and spinal fluid, amnioticfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder. Preferred polypeptides of thepresent invention comprise immunogenic epitopes shown in SEQ ID NO: 160as residues: Gly-35 to Asp-40, Asn-51 to Trp-59. Polynucleotidesencoding said polypeptides are also provided.

[0242] The tissue distribution in placenta indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and diagnosis of developmental anomalies or fetaldeficiencies, reproductive dysfunction, as well as ovarian and otherendometrial cancers.

[0243] Moreover, the expression within placenta and other cellularsources marked by proliferating cells indicates this protein may play arole in the regulation of cellular division, and may show utility in thediagnosis, treatment, and/or prevention of developmental diseases anddisorders, including cancer, and other proliferative conditions.Representative uses are described in the “Hyperproliferative Disorders”and “Regeneration” sections below and elsewhere herein. Briefly,developmental tissues rely on decisions involving cell differentiationand/or apoptosis in pattern formation. Dysregulation of apoptosis canresult in inappropriate suppression of cell death, as occurs in thedevelopment of some cancers, or in failure to control the extent of celldeath, as is believed to occur in acquired immunodeficiency and certaindegenerative disorders, such as spinal muscular atrophy (SMA). \

[0244] Alternatively, this gene product may be involved in the patternof cellular proliferation that accompanies early embryogenesis. Thus,aberrant expression of this gene product in tissues—particularly adulttissues—may correlate with patterns of abnormal cellular proliferation,such as found in various cancers. Because of potential roles inproliferation and differentiation, this gene product may haveapplications in the adult for tissue regeneration and the treatment ofcancers. It may also act as a morphogen to control cell and tissue typespecification. Therefore, the polynucleotides and polypeptides of thepresent invention are useful in treating, detecting, and/or preventingsaid disorders and conditions, in addition to other types ofdegenerative conditions. Thus this protein may modulate apoptosis ortissue differentiation and is useful in the detection, treatment, and/orprevention of degenerative or proliferative conditions and diseases. Theprotein is useful in modulating the immune response to aberrantpolypeptides, as may exist in proliferating and cancerous cells andtissues. The protein can also be used to gain new insight into theregulation of cellular growth and proliferation. Furthermore, theprotein may also be used to determine biological activity, raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0245] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:39 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1003 of SEQID NO:39, b is an integer of 15 to 1017, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:39, and whereb is greater than or equal to a +14.

[0246] Features of Protein Encoded by Gene No: 30

[0247] The translation product of this gene shares sequence homologywith ALS (Acid Labile Subunit of Insulin-Like Growth Factor) which isthought to be important in the regulation of IGF availability. As such,it is likely that the product of this gene is involved in the regulationof various proliferation-dependent cellular processes that may beattributable to cancer progression (See Genbank Accession No. gi|84808;all references available through this accession are hereby incorporatedby reference herein).

[0248] Preferred polypeptides of the invention comprise the followingamino acid sequence: FHGLGRLHTVHL (SEQ ID NO: 347),AAFTGLALLEQLDLSDNAQLR (SEQ ID NO: 348), HEVPDAPRPTPT (SEQ ID NO: 350),and/or AFRGLHSLD (SEQ ID NO: 349). Polynucleotides encoding thesepolypeptides are also provided.

[0249] The gene encoding the disclosed cDNA is believed to reside onchromosome 22. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 22.

[0250] This gene is expressed primarily in cerebellum and ovariancancer.

[0251] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,neurodegenerative diseases, growth deficiencies, osteoporosis, catabolicdisorders, diabetes, and ovarian cancer. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the nervous system and other peripheral tissues,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types (e.g., neural,proliferating, and/or other tissues) or bodily fluids (e.g., lymph,serum, plasma, urine, synovial fluid and spinal fluid) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 161 as residues: Thr41 toGly-47, Pro-170 to Asp-176, Leu-257 to Trp-262, Gln-276 to Ser-283,Arg-323 to Leu-330, Pro-362 to Val-374. Polynucleotides encoding saidpolypeptides are also provided.

[0252] The tissue distribution cerebellum and homology to ALS (AcidLabile Subunit of Insulin-Like Growth Factor) indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and diagnosis of a variety of metabolic disorders,growth deficiencies, osteoporosis, catabolic disorders (including AIDS)and diabetes. Nearly all of the insulin-like growth factor (IGF) in thecirculation is bound in a heterotrimeric complex composed of IGF,IGF-binding protein-3, and the acid-labile subunit (ALS). The proteinproduct of this gene therefore may afford the ability to potentiate thebiological actions of IGF or similar growth factors and cytokines.Studies which demonstrate the beneficial effect of IGF-I in amyotrophiclateral-sclerosis, would suggest a role in this disease as well.Alternatively, the tissue distribution in cancerous ovarian tissueindicates polynucleotides and polypeptides corresponding to this geneare useful for the treatment, diagnosis, and/or prevention of variousskin disorders. Furthermore, the protein may also be used to determinebiological activity, raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0253] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:40 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1763 of SEQID NO:40, b is an integer of 15 to 1777, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:40, and whereb is greater than or equal to a +14.

[0254] Features of Protein Encoded by Gene No: 31

[0255] The translation product of this gene was shown to have homologyto diacylglycerol kinase which is known to be important in lipidmetabolism (See Genbank Accession No.gi|1939; all references availablethrough this accession are hereby incorporated by reference herein).

[0256] Preferred polypeptides of the invention comprise the followingamino acid sequence:MVVADRNRASSSSYLCLLLFSLSLFLCHETVCDRATCLFFFLKFFFLFMCRCMSWGFKNFKAGLLMQSMPTSGILRERKRLHVVRIPQGTEKKLETVEMQI (SEQ ID NO: 351),and/or IPQGTEKKLETV (SEQ ID NO: 352). Polynucleotides encoding thesepolypeptides are also provided.

[0257] This gene is expressed primarily in brain.

[0258] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,developmental and neurodegenerative diseases of the brain and nervoussystem. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thebrain, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g.,neural, and/or other tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 162 as residues: Gly-49 toSer-54, Lys-61 to Arg-68. Polynucleotides encoding said polypeptides arealso provided.

[0259] The tissue distribution in brain combined with the homology to aknown enzyme involved in lipid metabolism indicates that polynucleotidesand polypeptides corresponding to this gene are useful for thedetection, treatment, and/or prevention of neurodegenerative diseasestates, behavioral disorders, or inflammatory conditions. Representativeuses are described in the “Regeneration” and “HyperproliferativeDisorders” sections below, in Example 11, 15, and 18, and elsewhereherein. Briefly, the uses include, but are not limited to the detection,treatment, and/or prevention of Alzheimer's Disease, Parkinson'sDisease, Huntington's Disease, Tourette Syndrome, meningitis,encephalitis, demyelinating diseases, peripheral neuropathies,neoplasia, trauma, congenital malformations, spinal cord injuries,ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania,dementia, paranoia, obsessive compulsive disorder, depression, panicdisorder, learning disabilities, ALS, psychoses, autism, and alteredbehaviors, including disorders in feeding, sleep patterns, balance, andperception. In particular, this gene may have utility in the diagnosis,treatment, and/or prevention of disorders involving the PNS, CNS and/orother tissues which rely on lipid-containing structures such as myelinsheath dependent nerves. Furthermore, the protein may also be used todetermine biological activity, to raise antibodies, as tissue markers,to isolate cognate ligands or receptors, to identify agents thatmodulate their interactions, in addition to its use as a nutritionalsupplement. Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tissues.

[0260] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:41 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 989 of SEQID NO:41, b is an integer of 15 to 1003, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:41, and whereb is greater than or equal to a +14.

[0261] Features of Protein Encoded by Gene No: 32

[0262] This gene is expressed primarily in amygdala and testes.

[0263] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,testicular disorders, and developmental and neurodegenerative diseasesof the brain and nervous system. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe brain, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO:163 as residues: Met-1 to Lys-6.Polynucleotides encoding said polypeptides are also provided.

[0264] The tissue distribution in amygdala indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the detection, treatment, and/or prevention of neurodegenerativedisease states, behavioral disorders, or inflammatory conditions.Representative uses are described in the “Regeneration” and“Hyperproliferative Disorders” sections below, in Example 11, 15, and18, and elsewhere herein. Briefly, the uses include, but are not limitedto the detection, treatment, and/or prevention of aphasia, depression,schizophrenia, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, specific brain tumors, mania, dementia, paranoia, addictivebehavior and sleep disorders. The amygdala processes sensory informationand relays this to other areas of the brain including the endocrine andautonomic domains of the hypothalamus and the brain stem. As such, thetranslation product of this gene may show commercial utility in thediagnosis, treatment, and/or prevention of various endocrine,cardiovascular, and pulmonary disorders, particularly those disordersdirectly associated with CNS/autonomic control. Furthermore, the proteinmay also be used to determine biological activity, to raise antibodies,as tissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0265] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:42 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1187 of SEQID NO:42, b is an integer of 15 to 1201, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:42, and whereb is greater than or equal to a +14.

[0266] Features of Protein Encoded by Gene No: 33

[0267] The gene encoding the disclosed cDNA is believed to reside onchromosome 9. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 9.

[0268] Preferred polypeptides of the invention comprise the followingamino acid sequence: NPRLPLPRGGSLRLLSSPANSNNAKAYPFSRFPSPIF (SEQ ID NO:353). Polynucleotides encoding these polypeptides are also provided.

[0269] This gene is expressed primarily in B-cell lymphoma.

[0270] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,haemopoietic and immune diseases and/or disorders including cancer.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of thehaemopoietic and immune system, expression of this gene at significantlyhigher or lower levels may be routinely detected in certain tissues orcell types (e.g., immune, hematopoietic, and cancerous and woundedtissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0271] The tissue distribution in B-cell lymphoma indicatespolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and diagnosis of hematopoietic related disorders suchas anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia sincestromal cells are important in the production of cells of hematopoieticlineages. Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Briefly, the uses include bone marrowcell ex-vivo culture, bone marrow transplantation, bone marrowreconstitution, radiotherapy or chemotherapy of neoplasia.

[0272] The gene product may also be involved in lymphopoiesis,therefore, it can be used in immune disorders such as infection,inflammation, allergy, immunodeficiency etc. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Therefore itis also useful as an agent for immunological disorders includingarthritis, asthma, immunodeficiency diseases such as AIDS, leukemia,rheumatoid arthritis, granulomatous disease, inflammatory bowel disease,sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities,such as T-cell mediated cytotoxicity; immune reactions to transplantedorgans and tissues, such as host-versus-graft and graft-versus-hostdiseases, or autoimmunity disorders, such as autoimmune infertility,lense tissue injury, demyelination, systemic lupus erythematosis, druginduced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, andscleroderma. Furthermore, the protein may also be used to determinebiological activity, to raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0273] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:43 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1162 of SEQID NO:43, b is an integer of 15 to 1176, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:43, and whereb is greater than or equal to a +14.

[0274] Features of Protein Encoded by Gene No: 34

[0275] This gene is expressed primarily in breast cancer.

[0276] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, diseasesand/or disorders of the reproductive organs and cancer, particularly ofthe mammary glands. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thereproductive system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., reproductive, breast, and cancerous and wounded tissues) orbodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder. Preferred polypeptides of thepresent invention comprise immunogenic epitopes shown in SEQ ID NO: 165as residues: Asp-77 to Gly-127. Polynucleotides encoding saidpolypeptides are also provided.

[0277] The tissue distribution in tumors of breast origins indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for diagnosis and intervention of such tumors, in addition toother tumors. Representative uses are described in the“Hyperproliferative Disorders”, “Infectious Disease”, and “BindingActivity” sections below, in Example 11, and 27, and elsewhere herein.Furthermore, the protein may also be used to determine biologicalactivity, raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0278] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:44 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 555 of SEQID NO:44, b is an integer of 15 to 569, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:44, and where bis greater than or equal to a +14.

[0279] Features of Protein Encoded by Gene No: 35

[0280] Preferred polypeptides encoded by this gene comprise thefollowing amino acid sequence:MVQEAPALVRLSLGSHRVKGPLPVLKLQPEGWSPSTLWSCASVWKDSC (SEQ ID NO: 354),and/or ALASSLVAENQGFVAALMVQEAPALVRLSLGSHRVKGPLPVLKLQPEGWSPSTLWSCASVWKDSCMHPWRLSMCPACVLAALPALCSCLCSPDARPPHGWMS MPFTPHPLVSRAMPTCHPCS(SEQ ID NO: 355). Polynucleotides encoding these polypeptides are alsoprovided.

[0281] The gene encoding the disclosed cDNA is believed to reside onchromosome 11. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 11.

[0282] This gene is expressed primarily in placenta, dendritic cells,brain, and to a lesser extent in infant cells and tissues.

[0283] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, diseasesand/or disorders of developing cells and tissues, particularly growthdisorders. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theplacenta and other developing organs and tissues, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., developing, neural, placental,brain, and cancerous and wounded tissues) or bodily fluids (e.g., lymph,serum, amniotic fluid, plasma, urine, synovial fluid and spinal fluid)or another tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder. Preferred polypeptides of the present inventioncomprise immunogenic epitopes shown in SEQ ID NO: 166 as residues:Pro-27 to Gly-34. Polynucleotides encoding said polypeptides are alsoprovided.

[0284] The tissue distribution in placental tissue indicates the proteinis useful in the detection, treatment, and/or prevention of vascularconditions, which include, but are not limited to, microvasculardisease, vascular leak syndrome, aneurysm, stroke, atherosclerosis,arteriosclerosis, or embolism. For example, this gene product mayrepresent a soluble factor produced by smooth muscle that regulates theinnervation of organs or regulates the survival of neighboring neurons.Likewise, it is involved in controlling the digestive process, and suchactions as peristalsis. Similarly, it is involved in controlling thevasculature in areas where smooth muscle surrounds the endothelium ofblood vessels. The expression within cellular sources marked byproliferating cells (e.g., infant cells and tissues) indicates thisprotein may play a role in the regulation of cellular division, and mayshow utility in the diagnosis, treatment, and/or prevention ofdevelopmental diseases and disorders, cancer, and other proliferativeconditions. Representative uses are described in the “HyperproliferativeDisorders” and “Regeneration” sections below and elsewhere herein.Briefly, developmental tissues rely on decisions involving celldifferentiation and/or apoptosis in pattern formation. Dysregulation ofapoptosis can result in inappropriate suppression of cell death, asoccurs in the development of some cancers, or in failure to control theextent of cell death, as is believed to occur in acquiredimmunodeficiency and certain neurodegenerative disorders, such as spinalmuscular atrophy (SMA). Because of potential roles in proliferation anddifferentiation, this gene product may have applications in the adultfor tissue regeneration and the treatment of cancers. It may also act asa morphogen to control cell and tissue type specification. Therefore,the polynucleotides and polypeptides of the present invention are usefulin treating, detecting, and/or preventing said disorders and conditions,in addition to other types of degenerative conditions. Thus this proteinmay modulate apoptosis or tissue differentiation and is useful in thedetection, treatment, and/or prevention of degenerative or proliferativeconditions and diseases. The protein is useful in modulating the immuneresponse to aberrant polypeptides, as may exist in proliferating andcancerous cells and tissues. The protein can also be used to gain newinsight into the regulation of cellular growth and proliferation.Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0285] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:45 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 972 of SEQID NO:45, b is an integer of 15 to 986, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:45, and where bis greater than or equal to a +14.

[0286] Features of Protein Encoded by Gene No: 36

[0287] The translation product of this gene shares sequence homologywith ion channel proteins which are thought to be important in manyphysiological processes including neural and muscular function (See, forexample, Genbank Accession No. gi|1065507, and gb|AAC68885.1; allreferences available through these accession numbers are herebyincorporated herein; for example, FEBS Lett. 445, 231-236 (1999)).Specifically, this protein is homologous to the putative four repeat ionchannel of Rattus norvegicus. Based upon the sequence similarity, thetranslation product of this gene is expected to share at least somebiological activities with ion channel proteins. Such activities areknown in the art, some of which are described elsewhere herein.Preferred polypeptides comprise the following amino acid sequence:FYFITLIFFLAWLVKNVFIAVIIETFAEIRVQF (SEQ ID NO: 356), SIFTVYEAASQEGWV (SEQID NO: 357), and/or HEGTSIFTVYEAASQEGWVFL (SEQ ID NO: 358). Alsopreferred are polynucleotides encoding these polypeptides.

[0288] This gene is expressed primarily in spinal cord.

[0289] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, diseasesof the central and peripheral nervous system, particularly neuraldegenerative conditions, and is useful in restoring cognitive function.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the neuralsystem, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g.,neural, brain, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder. Preferred polypeptides of the present inventioncomprise immunogenic epitopes shown in SEQ ID NO: 167 as residues: Phe-8to Ser-13, Ala-84 to Ser-90. Polynucleotides encoding said polypeptidesare also provided.

[0290] The tissue distribution in spinal cord tissue, combined with thehomology to ion channel proteins, indicates polynucleotides andpolypeptides corresponding to this gene are useful for the detection,treatment, and/or prevention of neurodegenerative disease states,behavioral disorders, or inflammatory conditions. Representative usesare described in the “Regeneration” and “Hyperproliferative Disorders”sections below, in Example 11, 15, and 18, and elsewhere herein.Briefly, the uses include, but are not limited to the detection,treatment, and/or prevention of Alzheimer's Disease, Parkinson'sDisease, Huntington's Disease, Tourette Syndrome, meningitis,encephalitis, demyelinating diseases, peripheral neuropathies,neoplasia, trauma, congenital malformations, spinal cord injuries,ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania,dementia, paranoia, obsessive compulsive disorder, depression, panicdisorder, learning disabilities, ALS, psychoses, autism, and alteredbehaviors, including disorders in feeding, sleep patterns, balance, andperception. In addition, elevated expression of this gene product inregions of the brain indicates it plays a role in normal neuralfunction. Potentially, this gene product is involved in synapseformation, neurotransmission, learning, cognition, homeostasis, orneuronal differentiation or survival. Furthermore, the protein may alsobe used to determine biological activity, to raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0291] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:46 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1526 of SEQID NO:46, b is an integer of 15 to 1540, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:46, and whereb is greater than or equal to a +14.

[0292] Features of Protein Encoded by Gene No: 37

[0293] When tested against fibroblast cell lines, supernatants removedfrom cells containing this gene activated the early growth response gene1 (EGR) pathway. Thus, it is likely that this gene activates fibroblastcells, and to a lesser extent, other cells and tissue cell-types,through the EGR signal transduction pathway. The early growth responsegene is a separate signal transduction pathway from the Jaks-STAT, genescontaining the EGR1 promoter are induced in various tissues and celltypes upon activation, leading the cells to undergo differentiation andproliferation. The polypeptide of this gene has been determined to havea transmembrane domain at about amino acid position 13-29 of the aminoacid sequence referenced in Table 1 for this gene. Moreover, acytoplasmic tail encompassing amino acids 30-59 of this protein has alsobeen determined. Based upon these characteristics, it is believed thatthe protein product of this gene shares structural features to type Ibmembrane proteins. Additionally, portions of the translation product ofthis gene shares sequence homology with TM5 consensus sequences (see,e.g., Genseq accession number R50725; all references available throughthis accession are hereby incorporated by reference herein.). Based onthe sequence similarity, the translation product of this gene isexpected to share at least some biological activities with TM5 proteins.Such activities are known in the art, some of which are describedelsewhere herein.

[0294] This gene is expressed primarily in uterus, colon cancer,synovium, fetal lung, and to a lesser extent in fetal and adult heart.

[0295] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, diseasesand/or disorders of developing cells and tissues, particularlyinfertility and cancer. Similarly, polypeptides and antibodies directedto these polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thedeveloping and reproductive systems, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., reproductive, developing,gastrointestinal, synovium, skeletal, heart, lung, cardiovascular, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,amniotic fluid, plasma, urine, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder. Preferred polypeptides of the present inventioncomprise immunogenic epitopes shown in SEQ ID NO: 168 as residues:Lys-32 to His-38. Polynucleotides encoding said polypeptides are alsoprovided.

[0296] The tissue distribution in developing and reproductive tissues,combined with the detected EGR1 biological activity, indicates thisprotein may play a role in the regulation of cellular division, and mayshow utility in the diagnosis, treatment, and/or prevention ofdevelopmental diseases and disorders, including cancer, and otherproliferative conditions. Representative uses are described in the“Hyperproliferative Disorders” and “Regeneration” sections below andelsewhere herein. Briefly, developmental tissues rely on decisionsinvolving cell differentiation and/or apoptosis in pattern formation.Dysregulation of apoptosis can result in inappropriate suppression ofcell death, as occurs in the development of some cancers, or in failureto control the extent of cell death, as is believed to occur in acquiredimmunodeficiency and certain neurodegenerative disorders, such as spinalmuscular atrophy (SMA). Because of potential roles in proliferation anddifferentiation, this gene product may have applications in the adultfor tissue regeneration and the treatment of cancers. It may also act asa morphogen to control cell and tissue type specification. Therefore,the polynucleotides and polypeptides of the present invention are usefulin treating, detecting, and/or preventing said disorders and conditions,in addition to certain types of degenerative conditions. Thus thisprotein may modulate apoptosis or tissue differentiation and is usefulin the detection, treatment, and/or prevention of degenerative orproliferative conditions and diseases. The protein is useful inmodulating the immune response to aberrant polypeptides, as may exist inproliferating and cancerous cells and tissues. The protein can also beused to gain new insight into the regulation of cellular growth andproliferation. The tissue distribution combined with the detected EGR1biological activity and homology to TM5 consensus regions indicates thisprotein likely plays a role in signal transduction and/or the regulationthereof, and may show utility in the diagnosis, treatment, and/orprevention of disorders in which the normal physiological signaltransduction pathway in disregulated. Furthermore, the protein may alsobe used to determine biological activity, to raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0297] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:47 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 778 of SEQID NO:47, b is an integer of 15 to 792, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:47, and where bis greater than or equal to a +14.

[0298] Features of Protein Encoded by Gene No: 38

[0299] Preferred polypeptides of the invention comprise the followingamino acid sequence: CKTSFGLA (SEQ ID NO: 359). Polynucleotides encodingthese polypeptides are also provided. In an alternative embodiment,polypeptides of the invention comprise the following amino acidsequence: MITLSSAFSAKQKTHAHKNTHACMCATDMANPKLVLHFEVIVALLSLLQTILSLLLGQRTWLAHLYVLSTENXALHTVGTQKHLLPHDWCFGKHCVSCRHHIFH RFCSIFSSTLKRSQGFEG(SEQ ID NO: 360). Polynucleotides encoding these polypeptides are alsoprovided.

[0300] This gene is expressed primarily in fetal bone, B and T celllymphoma, and dendritic cells.

[0301] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,hematopoietic, skeletal, and immune diseases and/or disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the immunesystem, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g.,immune, hematopoietic, skeletal, developmental, and cancerous andwounded tissues) or bodily fluids (e.g., lymph, serum, plasma, amnioticfluid, urine, synovial fluid and spinal fluid) or another tissue or cellsample taken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.Preferred polypeptides of the present invention comprise immunogenicepitopes shown in SEQ ID NO:169 as residues: Ser-33 to His-42.Polynucleotides encoding said polypeptides are also provided.

[0302] The tissue distribution in T-cells and dendritic cells indicatespolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and diagnosis of hematopoietic related disorders suchas anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia sincestromal cells are important in the production of cells of hematopoieticlineages. Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Briefly, the uses include bone marrowcell ex-vivo culture, bone marrow transplantation, bone marrowreconstitution, radiotherapy or chemotherapy of neoplasia.

[0303] The gene product may also be involved in lymphopoiesis,therefore, it can be used in immune disorders such as infection,inflammation, allergy, immunodeficiency etc. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types.

[0304] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0305] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:48 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1483 of SEQID NO:48, b is an integer of 15 to 1497, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:48, and whereb is greater than or equal to a +14.

[0306] Features of Protein Encoded by Gene No: 39

[0307] This gene is expressed primarily in prostate.

[0308] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,reproductive diseases and/or disorders, particularly prostate cancer.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the malereproductive system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., reproductive, prostate, and cancerous and wounded tissues) orbodily fluids (e.g., lymph, serum, plasma, urine, seminal fluid,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO: 170 as residues: Pro-21 to Pro-26, Arg-31 to Asn-37. Polynucleotidesencoding said polypeptides are also provided.

[0309] The tissue distribution in prostate tissue indicates that theprotein products of this gene are useful for the diagnosis andintervention of prostate cancers, in addition to other tumors within theurogenital and reproductive system. Therefore, this gene product isuseful in the treatment of male infertility and/or impotence. This geneproduct is also useful in assays designed to identify binding agents, assuch agents (antagonists) are useful as male contraceptive agents.Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0310] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:49 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1326 of SEQID NO:49, b is an integer of 15 to 1340, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:49, and whereb is greater than or equal to a +14.

[0311] Features of Protein Encoded by Gene No: 40

[0312] The translation product of this gene shares sequence homologywith the human proliferating-cell nucleolar antigen as well as to aprotein from Schizosaccharomyces pombe of unknown function (See GenbankAccession Nos. 189422 and gnl|PID|e349594, as well as Medline Article90315275; all references available through these accessions are herebyincorporated herein by reference). This protein is the most cancerspecific of the proliferation-associated nucleolar proteins identifiedthus far. In addition, it is of special interest because of itsexpression pattern in the early G1 phase, and, in studies prior to 1989,it has not been detected in benign tumors and most normal restingtissues.

[0313] In another embodiment, polypeptides of the invention comprise thefollowing amino acid sequence:SATEHGAVCCSCRRVGRRGEPPGSIKGLVYSSNFQNVKQLYALVCETQRYSAVLDAVIASAGLLRAEKKLRPHLAKVLVYELLLGKGFRGGGGRWKALLGRHQARLKAELARLKVHRGVSRNEDLLEVGSRPGPASQLPRFVRVNTLKTCSDDVVDYFKRQGFSYQGRASSLDDLRALKGKHFLLDPLMPELLVFPAQTDLHEHPLYRAGHLILQDRASCLPAMLLDPPPGSHVIDACAAPGNKTSHLAALLKNQGKIFAFDLDAKRLASMATLLAXAGVSCCELAEEDFLAVSPXDPRYXEVHYXLLDPSCSGSGMPSRQLEXPGAGTPSPVRLHALAGFQQRALCHALTFPSLQRLVYSTCSLCQEENEDVVRDALQQNPGAFRLAPALPAWPHRGLSTFPGAEHCLRASPETTLSSGFFVAVIERVEXPSSASQAKASAPERTPSPAPKRKKRQQRAAAGACTPPCT (SEQ ID NO: 365),CAAPGNKTSHLAA (SEQ ID NO: 361), EHPLYRAGHLILQDRASCLPAMLL (SEQ ID NO:362), LLDPSCSGSGMPSRQ (SEQ ID NO: 363), YSTCSLCQEENEDVVRDALQQNP (SEQ IDNO: 364), and/or YEPHSTHSRERAMTSHARVSLGPSRDPLERPHLAKVLVYELLLGKGFRGGGGRWKALLGRHQARLKAELARLKVHRGVSRNEDLLEVGSRPGPASQLPRFVRVNTLKTCSDDVVDYFKRQGFSYQGRASSLDDLRALKGKHFLLDPLMPELLVFPAQTDLHEHPLYRAGHLILQDRASCLPAMLLDPPPGSHVIDACAAPGNKTSHL AALLKNQGKIFAFDLDAKRLASMATLLAXAGVSCCELAEEDFLAVSPXDPRYXEVHYXLLDPSCSGSGMPSRQLEEPGAGTPSPVRLHALAGFQQRALCHALTFPSLQRLVYSTCSLCQEENEDVVRDALQQNPGAFRLAPALPAWPHRGLSTFPGAEHCLRASPETTLSSGFFVAVIERVEVPSSASQAKASAPERTPSPAPKRKKRQQXAA AGACTPPCT (SEQ IDNO: 366). Polynucleotides encoding these polypeptides are also provided.This gene maps to chromosome 7, and therefore, may be used as a markerin linkage analysis for chromosome 7.

[0314] This gene is expressed primarily in T cells and rejected kidneyand to a lesser extent in keratinocytes and various other normal andtransformed, predominately haemopoietic cell types.

[0315] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immunediseases and/or disorders, particularly host-vs-graft disease, andtransplant rejection. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,rejected transplant tissue, immune, heamatopoietic, and cancerous andwounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0316] The tissue distribution in T-cells and rejected kidney, indicatespolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Briefly, the expression of this geneproduct indicates a role in regulating the proliferation; survival;differentiation; and/or activation of hematopoietic cell lineages,including blood stem cells. Involvement in the regulation of cytokineproduction, antigen presentation, or other processes suggests ausefulness in the treatment of cancer (e.g. by boosting immuneresponses). Expression in cells of lymphoid origin, the natural geneproduct would be involved in immune functions. Therefore it is alsouseful as an agent for immunological disorders including arthritis,asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoidarthritis, granulomatous disease, inflammatory bowel disease, sepsis,acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such asT-cell mediated cytotoxicity; immune reactions to transplanted organsand tissues, such as host-versus-graft and graft-versus-host diseases,or autoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, andscleroderma.

[0317] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. Thus, this gene productis thought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0318] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:50 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1525 of SEQID NO:50, b is an integer of 15 to 1539, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:50, and whereb is greater than or equal to a +14.

[0319] Features of Protein Encoded by Gene No: 41

[0320] The gene encoding the disclosed cDNA is believed to reside onchromosome 12. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 12.

[0321] This gene is expressed primarily in placenta, uterus, 12 weekold, early stage, embryo and to a lesser extent in epithelium.

[0322] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,developmental and reproductive diseases and/or disorders, in addition todisorders of the integumentary system. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the developmental and epithelial tissues, expression ofthis gene at significantly higher or lower levels may be routinelydetected in certain tissues or cell types (e.g., developmental,reproductive, uterine, placental, integumentary, and cancerous andwounded tissues) or bodily fluids (e.g., lymph, serum, plasma, amnioticfluid, urine, synovial fluid and spinal fluid) or another tissue or cellsample taken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.

[0323] The tissue distribution in placental, uterine, and embryoniccells and tissues indicates this protein may play a role in theregulation of cellular division, and may show utility in the diagnosis,treatment, and/or prevention of developmental diseases and disorders,including cancer, and other proliferative conditions. Representativeuses are described in the “Hyperproliferative Disorders” and“Regeneration” sections below and elsewhere herein. Briefly,developmental tissues rely on decisions involving cell differentiationand/or apoptosis in pattern formation. Dysregulation of apoptosis canresult in inappropriate suppression of cell death, as occurs in thedevelopment of some cancers, or in failure to control the extent of celldeath, as is believed to occur in acquired immunodeficiency and certainneurodegenerative disorders, such as spinal muscular atrophy (SMA).Because of potential roles in proliferation and differentiation, thisgene product may have applications in the adult for tissue regenerationand the treatment of cancers. It may also act as a morphogen to controlcell and tissue type specification. Therefore, the polynucleotides andpolypeptides of the present invention are useful in treating, detecting,and/or preventing said disorders and conditions, in addition to othertypes of degenerative conditions. Thus this protein may modulateapoptosis or tissue differentiation and is useful in the detection,treatment, and/or prevention of degenerative or proliferative conditionsand diseases. The protein is useful in modulating the immune response toaberrant polypeptides, as may exist in proliferating and cancerous cellsand tissues. The protein can also be used to gain new insight into theregulation of cellular growth and proliferation. The protein may beuseful for the detection, treatment, and/or prevention of various typesof cancer, particularly of the integumentary system. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0324] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:51 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1409 of SEQID NO:51, b is an integer of 15 to 1423, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:51, and whereb is greater than or equal to a +14.

[0325] Features of Protein Encoded by Gene No: 42

[0326] The translation product of this gene was shown to have homologyto the human, bovine, mouse, and rat G protein gamma-3 subunit (SeeGenbank Accession Nos.WO9413, pir|A36204|RGBOG3, gi|2582400 (AF022088),and gi|1353498) which are known to play a role in the regulation ofsignal transduction pathways.

[0327] Moreover, the protein shares structural homology to a yeastmitochondrion membrane protein Q0225 (See Genbank Accession No.pir|S72689|S72689).

[0328] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: NREQKAKSQLLRSQLYSTIDLPYFFQCVGTRCTAVCVCVCVCVCVCXYLPIHWQVNLHLVYLAMLCFLPIPLLSILSPQTQASRLLDETVRRKHFLTYPFGISSIIT QALL (SEQ ID NO:369). Polynucleotides encoding these polypeptides are also provided.

[0329] In yet another embodiment, polypeptides of the invention comprisethe following amino acid sequence: MGTHSVSGRFSKTSPPYCPPSSSLPGPISSIGFNKS(SEQ ID NO: 367)LHECLFISEKELLPLPFPFPDLKSFISYLTSMLKPGPLIVSLKIWVSYPITRPRYLPPMLKSLNISFLYIQYIWAYIHLYTSFYIYIISVSFFLDKPFIYVISFPKPPHFLFASLSKTQEFHFHVPQHHFFLIFSPQVSSPISCFARLLKSPLFTPVPTEISPFYNCAYYSADIPSPQLVWGPISHQTWLLLKLGLLPKRGFQVRGD RL, and/orCFARLLKSPLFTPVPTEISPFYNCAYYSA. (SEQ ID NO: 368)

[0330] Polynucleotides encoding these polypeptides are also provided.

[0331] This gene is expressed primarily in infant brain, fetal tissue,frontal cortex, corpus collosum, and to a lesser extent in amygdalatissue.

[0332] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, neuraland CNS diseases and/or disorders. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the central nervous and peripheral nervous systems,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types (e.g., neural, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO:173 as residues: Thr-26 toLeu-33. Polynucleotides encoding said polypeptides are also provided.

[0333] The tissue distribution in various neural cells and tissues,combined with the similarity to G Protein Gamma-3 subunit indicatespolynucleotides and polypeptides corresponding to this gene are usefulfor the detection, treatment, and/or prevention of neurodegenerativedisease states, behavioral disorders, or inflammatory conditions.Representative uses are described in the “Regeneration” and“Hyperproliferative Disorders” sections below, in Example 11, 15, and18, and elsewhere herein. Briefly, the uses include, but are not limitedto the detection, treatment, and/or prevention of Alzheimer's Disease,Parkinson's Disease, Huntington's Disease, Tourette Syndrome,meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0334] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:52 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1350 of SEQID NO:52, b is an integer of 15 to 1364, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:52, and whereb is greater than or equal to a +14.

[0335] Features of Protein Encoded by Gene No: 43

[0336] The translation product of this gene shares homology with thehuman alpha-3 type IX collagen protein (See Genbank AccessionNo.gi|196421) and mitsugumin 23, a novel transmembrane protein onendoplasmic reticulum and nuclear membranes (e.g., Genbank accessionnumber BAA33366; all references available through this accession arehereby incorporated by reference herein.) This protein likely representsa Type IIIb membrane protein. Although the preferred open reading frameof the present invention contains a signal peptide (as delineated inTable 1 and described elsewhere herein), the protein appears to haveseveral transmembrane domains. The transmembrane domains are located atabout amino acid position 111-162, 137-162, 163-186, and 64-85 of thesequence referenced in Table 1 for this gene. Preferred are polypeptidescomprising the following amino acid sequence:PGPEAQPWPGPDLPAVGSRGPGRLLAAVSAPRLGLGLAGADPVGPEACHLP (SEQ ID NO: 370),GRLRGPDEVGAPFHPGPATPGLADPLRPAEPXHWLPSLWGPT (SEQ ID NO: 371),PGPEAQPWPGPDLPAVGSR (SEQ ID NO: 372), and/or ATPGLADPLRPAEPXHWLP (SEQ IDNO: 373). Polynucleotides encoding these polypeptides are also provided.

[0337] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: QWPEKDPVMAASSISSPWGKHVFKAILMVLVALILLHSALAQSRRDFAPPGQQKREAPVDVLTQIGRSVRGTLDAWIGPETMHLVSESSSQVLWAISSAISVAFFALSGIAAQLLNALGLAGDYLAQGLKLSPGQVQTFLLWGAGALVVYWLLSLLLGLVLALLGRILWGLKLVIFLAGFVALMRSVPDPSTRALLLLALLILYALLSRXTGSRASGAQLEAKVRGLERQVEELRWRQRQXAKGARSVEEE (SEQ ID NO: 374).Polynucleotides encoding these polypeptides are also provided.

[0338] The gene encoding the disclosed cDNA is believed to reside onchromosome 11. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 11.

[0339] This gene is expressed primarily in melanocytes, and to a lesserextent in synovial sarcoma and larynx sarcoma.

[0340] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, melanomaand other disorders of the integumentary system. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the synovial and epithelial tissues, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., integumentary, and cancerous andwounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO:174 as residues: Gln-15 to Phe-20, Pro-22 to Ala-30, Val-160 toThr-165. Polynucleotides encoding said polypeptides are also provided.

[0341] The tissue distribution in melanocytes and sarcoma tissueindicates that polynucleotides and polypeptides corresponding to thisgene are useful for the study treatment and diagnosis of various cancersand their metastases, particularly of the integumentary system.Additionally, the homology to a conserved collagen protein would suggestthat this protein may also be important in the diagnosis or treatment ofvarious autoimmune disorders such as rheumatoid arthritis, lupus,scleroderma, and dermatomyositis as well as dwarfism, spinaldeformation, and specific joint abnormalities as well aschondrodysplasias ie. spondyloepiphyseal dysplasia congenita, familialosteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasiatype Schmid.

[0342] Moreover, polynucleotides and polypeptides corresponding to thisgene are useful for the treatment, diagnosis, and/or prevention ofvarious skin disorders. Representative uses are described in the“Biological Activity”, “Hyperproliferative Disorders”, “InfectiousDisease”, and “Regeneration” sections below, in Example 11, 19, and 20,and elsewhere herein. Briefly, the protein is useful in detecting,treating, and/or preventing congenital disorders (i.e. nevi, moles,freckles, Mongolian spots, hemangiomas, port-wine syndrome),integumentary tumors (i.e. keratoses, Bowen's disease, basal cellcarcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease,mycosis fungoides, and Kaposi's sarcoma), injuries and inflammation ofthe skin (i.e. wounds, rashes, prickly heat disorder, psoriasis,dermatitis), atherosclerosis, uticaria, eczema, photosensitivity,autoimmune disorders (i.e. lupus erythematosus, vitiligo,dermatomyositis, morphea, scleroderma, pemphigoid, and pemphigus),keloids, striae, erythema, petechiae, purpura, and xanthelasma. Inaddition, such disorders may predispose increased susceptibility toviral and bacterial infections of the skin (i.e. cold sores, warts,chickenpox, molluscum contagiosum, herpes zoster, boils, cellulitis,erysipelas, impetigo, tinea, althlete's foot, and ringworm).

[0343] Moreover, the protein product of this gene may also be useful forthe treatment or diagnosis of various connective tissue disorders (i.e.,arthritis, trauma, tendonitis, chrondomalacia and inflammation, etc.),autoimmune disorders (i.e., rheumatoid arthritis, lupus, scleroderma,dermatomyositis, etc.), dwarfism, spinal deformation, jointabnormalities, and chondrodysplasias (i.e. spondyloepiphyseal dysplasiacongenita, familial osteoarthritis, Atelosteogenesis type II,metaphyseal chondrodysplasia type Schmid). Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0344] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:53 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 2274 of SEQID NO:53, b is an integer of 15 to 2288, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:53, and whereb is greater than or equal to a +14.

[0345] Features of Protein Encoded by Gene No: 44

[0346] The translation product of this gene shares sequence homologywith tumor progression inhibitor which is thought to be important ininhibition of tumor growth as well as its metastasis (See GenbankAccession No. W26667; all references available through this accessionare hereby incorporated by reference herein). The translation product ofthis gene also shares sequence homology with melastatin 1 (see, e.g.,Genbank accession number AAC80000; all references available through thisaccession are hereby incorporated by reference herein.) whose expressionis inversely correlated with melanoma aggressiveness. Based on thesequence similarity, the translation product of this gene is expected toshare at least some biological activities with melastatin proteins. Suchactivities are known in the art, some of which are described elsewhereherein. Preferred are polypeptides comprising the following amino acidsequence: EXPRXIXGXNAPQVPVRNSRVDPRVRPRVRSLVFVLFCDEVRQWYVNGVNYFTDLWNVMDTLGLFYFIAGIVFRLHSSNKSSLYSGRVIFCLDYIIFTLRLIHIFTV SRNLGPKII (SEQID NO: 375), NILLVNLLVAMF (SEQ ID NO: 376), and/or QVWKFQRYFL (SEQ IDNO: 377). Polynucleotides encoding these polypeptides are also provided.

[0347] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: EXPRXIXGXNAPQVPVRNSRVDPRVRPRVRSLVFVLFCDEVRQWYVNGVNY (SEQ IDNO: 378) FTDLWNVMDTLGLFYFIAGIVFRLHSSNKSSLYSGRVIFCLDYIIFTLRLIHIFTVSRNLGPKIIMLQRMLIDVXXFLFLFAVWMVAFGVAXQGILRQNEQRWRWIFRSVIYEPXLAMFGQVPSXVDGTTYDFAHCTFTGNESKPLCVXLDEHNLPRFPEWITIPLVCIYMLSTNILLVNLLVAMFGYTVGTVQENNDQVWKFQRYFLVQEY CSRLNIPFPFIVFAYFYMVVKKCFKCCCKEXNXESSVCCSKMXTMRLWHGRVS.

[0348] Polynucleotides encoding these polypeptides are also provided.

[0349] This gene is expressed primarily in adult liver, prostate, gallbladder, and to a lesser extent, in Hodkin's lymphoma II.

[0350] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, livercancer and other hepatic diseases and/or disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the liver, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., hepatic, reproductive, metabolic,immune, hematopoietic, and cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, bile, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0351] The tissue distribution in liver and gall bladder cells andtissues indicates polynucleotides and polypeptides corresponding to thisgene are useful for the detection and treatment of liver disorders andcancers. Representative uses are described in the “HyperproliferativeDisorders”, “Infectious Disease”, and “Binding Activity” sections below,in Example 11, and 27, and elsewhere herein. Briefly, the protein can beused for the detection, treatment, and/or prevention of hepatoblastoma,jaundice, hepatitis, liver metabolic diseases and conditions that areattributable to the differentiation of hepatocyte progenitor cells. Inaddition, this gene product may have commercial utility in the expansionof stem cells and committed progenitors of various blood lineages, andin the differentiation and/or proliferation of various cell types.Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0352] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:54 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1498 of SEQID NO:54, b is an integer of 15 to 1512, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:54, and whereb is greater than or equal to a +14.

[0353] Features of Protein Encoded by Gene No: 45

[0354] The polypeptide of the present invention is thought to have anintramitochondrial signal indicating that the protein could play a rolein metabolic processes, including apoptosis. Based upon this fact, it isexpected that the protein product of this gene will share at least somebiological activities with other mitochondrial proteins having a similarsignal. Such activities are known in the art, some of which aredescribed elsewhere.

[0355] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: MEFQNMYIQLFGFSFIVIIVRMLLLGLCVS ARQPVMPRATLWGHLSPAWVLVPWTPRACGQAAPGRGHVASDHKSGLPWPKHCSCLHPRASQPCLFSLNSNRTVFTAIQRVALGWTFWVQANLVPRCT (SEQ ID NO: 379). Polynucleotides encodingthese polypeptides are also provided.

[0356] The gene encoding the disclosed cDNA is believed to reside onchromosome 4. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 4.

[0357] This gene is expressed primarily in human prostate cancer, and toa lesser extent in soares melanocyte and human colon.

[0358] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, prostatecancer, melanoma, and other diseases and/or disorders of theintegumentary system. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of themale reproductive system, expression of this gene at significantlyhigher or lower levels may be routinely detected in certain tissues orcell types (e.g., prostate, reproductive, intregumentary, and cancerousand wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,seminal fluid, urine, synovial fluid and spinal fluid) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO:176 as residues: Ser-36 toGly-41, Pro-43 to Ser-49. Polynucleotides encoding said polypeptides arealso provided.

[0359] The tissue distribution in tumors of prostate, colon, andintegument origins indicates that polynucleotides and polypeptidescorresponding to this gene are useful for diagnosis and intervention ofthese tumors, in addition to other tumors where expression has beenindicated. Representative uses are described elsewhere herein. Briefly,the uses include, but are not limited to the detection, treatment,and/or prevention of male infertility and/or impotence. This geneproduct is also useful in assays designed to identify binding agents, assuch agents (antagonists) are useful as male contraceptive agents.Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0360] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:55 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1343 of SEQID NO:55, b is an integer of 15 to 1357, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:55, and whereb is greater than or equal to a +14.

[0361] Features of Protein Encoded by Gene No: 46

[0362] The gene encoding the disclosed cDNA is believed to reside onchromosome 17. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 17.

[0363] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: LLLCVTGVYSYGLMHPIPSSFMIKAVSSFLTAEEASVGNPEGAFMKVLQARKNXTSTELIVEPEEPSDSSGINLSGFGSEQLDTNDESDXISTLSYILPYFSAVNLDVXSXLLPFIKLPTXGNSLAKIQTVGQNXQXVXRVLMGPRSIQKRHFKEVGRQSIRREQGAQASVENAAEEKRLGSPAPREXEQPHTQQGPEKLAGNAXYTKPSFTQEHKAAVSVLXPFSKGAPSTSSPAKALPQVRDRWKDXTHXISILESAKARVTNMKASKPISHSRKKYRFHKTRSRMTHRTPKVKKSPKFRKKSYLSRLMLANRPPFSAAXSLINSPSQGAFSSLGDLSPQENPFLXVSAPSEHFIETTNIKDTTARNALEENVFMENTNMPEVTISENTNYNHPPEADSXGTAFNLGPTVKQTET (SEQ ID NO: 380).Polynucleotides encoding these polypeptides are also provided.

[0364] This gene is expressed primarily in brain, duodenum carcinoma andcheek carcinoma.

[0365] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,gastrointestinal disorders and carcinomas, in addition to disorders ofthe epithelium and mucosa. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe digestive system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., gastrointestinal, epithelial, mucosa, and cancerous and woundedtissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0366] The tissue distribution in duodenal tissues and epitheliaindicates that the protein product of this gene may be useful for thediagnosis and intervention of tumors and other disorders within thesetissues, in addition to other tumors. The expression within embryonictissue and other cellular sources marked by proliferating cellsindicates this protein may play a role in the regulation of cellulardivision, and may show utility in the diagnosis, treatment, and/orprevention of developmental diseases and disorders, including cancer,and other proliferative conditions. Representative uses are described inthe “Hyperproliferative Disorders” and “Regeneration” sections below andelsewhere herein. Briefly, this protein may modulate apoptosis or tissuedifferentiation and is useful in the detection, treatment, and/orprevention of degenerative or proliferative conditions and diseases. Theprotein is useful in modulating the immune response to aberrantpolypeptides, as may exist in proliferating and cancerous cells andtissues. The protein can also be used to gain new insight into theregulation of cellular growth and proliferation. The tissue distributionin brain indicates polynucleotides and polypeptides corresponding tothis gene are useful for the detection, treatment, and/or prevention ofneurodegenerative disease states, behavioral disorders, or inflammatoryconditions. Representative uses are described in the “Regeneration” and“Hyperproliferative Disorders” sections below, in Example 11, 15, and18, and elsewhere herein. Briefly, the uses include, but are not limitedto the detection, treatment, and/or prevention of Alzheimer's Disease,Parkinson's Disease, Huntington's Disease, Tourette Syndrome,meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0367] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:56 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1975 of SEQID NO:56, b is an integer of 15 to 1989, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:56, and whereb is greater than or equal to a +14.

[0368] Features of Protein Encoded by Gene No: 47

[0369] The translation product of this gene shares sequence homologywith mouse magnesium dependent protein phosphatase (See GenbankAccession Nos. gnl|PID|d1004752 and emb|CAA06555.1| (AJ005458); allreferences available through these accessions are hereby incorporatedherein by reference; for example, J. Neurosci. Res. 51 (3), 328-338(1998)) which is thought to be important in normal protein metabolismand possibly gene regulation. Based on the sequence similarity, thetranslation product of this gene is expected to share at least somebiological activities with phosphatase proteins. Such activities areknown in the art, some of which are described elsewhere herein.Preferred polypeptides comprise the following amino acid sequence:CFSNAPKVSDEAVKKDSELDKHLESRVEEIMEKSGEEGMPDLAHVMRILSAE NIPNLPPGGGLAGXRNVIEAVYSRLNPHRESDGGAGDLEDPW (SEQ ID NO: 381),CFSNAPKVSDEAVKKDSELDKHLESRVEEIMEKSGEEGMPDLAHVMRILSAE NIPN (SEQ ID NO:382), RNVIEAVYSRLNPHRESDGGAGDLED (SEQ ID NO: 383), DSELDKHLESRVEEIM (SEQID NO: 384), KSGEEGMPDLAHVMRILSAENIPN (SEQ ID NO: 385), and/or CFSNAPKVS(SEQ ID NO: 386). Polynucleotides encoding these polypeptides are alsoprovided. A preferred polypeptide fragment of the invention comprisesthe following amino acid sequence:MSRKSLAFPIICSYLCFLTVATCSIACTTVFFANLRHTRYICIELSALETSGVISP QINNVPEVHGKYS(SEQ ID NO: 387). Polynucleotides encoding these polypeptides are alsoprovided.

[0370] This gene is expressed primarily in prostate and to a lesserextent in melanocytes.

[0371] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,proliferative conditions and cancers, in addition to reproductive,visual, and integumentary diseases and/or disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the reproductive system, expression ofthis gene at significantly higher or lower levels may be routinelydetected in certain tissues or cell types (e.g., reproductive, visual,retinal, integumentary, and cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, aqueous humor, vitreoushumor, synovial fluid and spinal fluid) or another tissue or cell sampletaken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.Preferred polypeptides of the present invention comprise immunogenicepitopes shown in SEQ ID NO: 178 as residues: Asp-6 to His-13, Asp-114to Gly-131, Thr-166 to Gln-181, Val-210 to Thr-216, Pro-222 to Tyr-227.Polynucleotides encoding said polypeptides are also provided.

[0372] The tissue distribution in prostate tissue, combined with thehomology to mouse magnesium dependent protein phosphatase indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the study and treatment of various cancers and reproductivedisorders. This protein may play a role in the regulation of cellulardivision, and may show utility in the diagnosis, treatment, and/orprevention of developmental diseases and disorders, including cancer,and other proliferative conditions. Representative uses are described inthe “Hyperproliferative Disorders” and “Regeneration” sections below andelsewhere herein. Briefly, developmental tissues rely on decisionsinvolving cell differentiation and/or apoptosis in pattern formation.Dysregulation of apoptosis can result in inappropriate suppression ofcell death, as occurs in the development of some cancers, or in failureto control the extent of cell death, as is believed to occur in acquiredimmunodeficiency and certain neurodegenerative disorders, such as spinalmuscular atrophy (SMA). This protein may modulate apoptosis or tissuedifferentiation and is useful in the detection, treatment, and/orprevention of degenerative or proliferative conditions and diseases. Theprotein is useful in modulating the immune response to aberrantpolypeptides, as may exist in proliferating and cancerous cells andtissues. The protein can also be used to gain new insight into theregulation of cellular growth and proliferation. The activity of thisprotein has been determined to be dependent upon the presence ofmagnesium ions. This protein is useful in the treatment, detection,and/or prevention of various visual disorders, particularly degenerativeconditions, and retinitis pigmentosa. Furthermore, the protein may alsobe used to determine biological activity, to raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0373] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:57 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 2529 of SEQID NO:57, b is an integer of 15 to 2543, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:57, and whereb is greater than or equal to a +14.

[0374] Features of Protein Encoded by Gene No: 48

[0375] The translation product of this gene shares sequence homologywith ribosomal protein L32 and L14, a mitochondrial protein from rattissues thought to be important in translation (See Genbank AccessionNo.gi|868267). Preferred are polypeptides comprising the following aminoacid sequence: IQKMTRVRVVDNSALG (SEQ ID NO: 388), PRCIHVYKKNGVGK (SEQ IDNO: 389), GDQILLAIKGQKKKA (SEQ ID NO: 390), and/or NPVGTRIKTPIPTSL (SEQID NO: 391). Polynucleotides encoding these polypeptides are alsoprovided.

[0376] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: VLIPSFSSSFLCSRGGPLPXDLSWDPMAFFTGLWGPFTCVSRVLSHHCFSTTGS (SEQ IDNO: 392) LSAIQKMTRVRVVDNSALGNSPYHRAPRCIHVYKKNGVGKVGDQILLAIKGQKKKALIVGHCMPGPRMTPRFDSNNVVLIED NGNPVGTRIKTPIPTSLRKREGEYSKVLAIAQNFV.

[0377] Polynucleotides encoding these polypeptides are also provided.This gene maps to chromosome 6, and therefore, may be used as a markerin linkage analysis for chromosome 6.

[0378] This gene is expressed primarily in uterus, fetal liver/spleen,human endometrial stromal cells-treated with estradiol and amnioticcells—Primary Culture, and to a lesser extent in, human fetal kidney.

[0379] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,endometriosis and reproductive disorders, particularly of the femalereproductive system. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thefemale reproductive system, expression of this gene at significantlyhigher or lower levels may be routinely detected in certain tissues orcell types (e.g., uterine, endometrium, reproductive, immune,hematopoietic, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder. Preferred polypeptides of the present inventioncomprise immunogenic epitopes shown in SEQ ID NO: 179 as residues:Pro-92 to Ser-102, Leu-127 to Tyr-134. Polynucleotides encoding saidpolypeptides are also provided.

[0380] The tissue distribution in endometrium and uterine tissues,combined with the homology to a ribosomal protein indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor diagnosis and intervention of tumors within said tissue, in additionto other tumors where expression has been indicated. This protein mayplay a role in cellular division, and may show utility in the diagnosis,treatment, and/or prevention of developmental diseases and disorders,including cancer, and other proliferative conditions. Representativeuses are described in the “Hyperproliferative Disorders” and“Regeneration” sections below and elsewhere herein. Briefly,developmental tissues rely on decisions involving cell differentiationand/or apoptosis in pattern formation. Dysregulation of apoptosis canresult in inappropriate suppression of cell death, as occurs in thedevelopment of some cancers, or in failure to control the extent of celldeath, as is believed to occur in acquired immunodeficiency and certainneurodegenerative disorders, such as spinal muscular atrophy (SMA).Because of potential roles in proliferation and differentiation, thisgene product may have applications in the adult for tissue regenerationand the treatment of cancers. It may also act as a morphogen to controlcell and tissue type specification. Therefore, the polynucleotides andpolypeptides of the present invention are useful in treating, detecting,and/or preventing said disorders and conditions, in addition to othertypes of degenerative conditions. Antagonists, including antibodiesdirected against this invention, is useful in inhibiting cellularproliferation and thus is useful in inhibiting cancers, in addition toother proliferative diseases and/or disorders. The protein is useful inmodulating the immune response to aberrant polypeptides, as may exist inproliferating and cancerous cells and tissues. The protein can also beused to gain new insight into the regulation of cellular growth andproliferation. Furthermore, the protein may also be used to determinebiological activity, to raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0381] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:58 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 763 of SEQID NO:58, b is an integer of 15 to 777, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:58, and where bis greater than or equal to a +14.

[0382] Features of Protein Encoded by Gene No: 49

[0383] This gene is expressed primarily in liver, hepatoma and to alesser extent in epithelial-TNFa and INF induced.

[0384] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, liverdiseases and/or disorders, particularly cancer. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the hepatic system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., hepatic, liver, and cancerous andwounded tissues) or bodily fluids (e.g., lymph, bile, serum, plasma,urine, synovial fluid and spinal fluid) or another tissue or cell sampletaken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.Preferred polypeptides of the present invention comprise immunogenicepitopes shown in SEQ ID NO: 180 as residues: Glu-28 to Gly-45, Ser-63to Gly-69, Gln-96 to Trp-104, Gly-112 to Pro-117, Arg-121 to Pro-128.Polynucleotides encoding said polypeptides are also provided.

[0385] The tissue distribution in liver and hepatoma tissue indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for the detection and treatment of liver disorders and cancers(e.g. hepatoblastoma, jaundice, hepatitis, liver metabolic diseases andconditions that are attributable to the differentiation of hepatocyteprogenitor cells). Representative uses are described in the“Hyperproliferative Disorders”, “Infectious Disease”, and “BindingActivity” sections below, in Example 11, and 27, and elsewhere herein.The protein is useful in modulating the immune response to aberrantpolypeptides, as may exist in proliferating and cancerous cells andtissues. The protein can also be used to gain new insight into theregulation of cellular growth and proliferation. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0386] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:59 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 865 of SEQID NO:59, b is an integer of 15 to 879, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:59, and where bis greater than or equal to a +14.

[0387] Features of Protein Encoded by Gene No: 50

[0388] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: ARVVQPAARAGMWAGGRSSCQAEVLRATRGGAARGNAAPGRALEMVPGAAGWCCLVLWLPACVAAHGFRIHDYLYFQVLSPGDIRYIFTATPAKDFGGIFHTRYEQIHLVPAEPPEACGELSNGFFIQDQIALVERGGCSFLSKTRVVQEHGGRA VIISDNALTMTASTWR(SEQ ID NO: 393). Polynucleotides encoding these polypeptides are alsoprovided.

[0389] In another embodiment, polypeptides comprising amino acidsequences of alternate downstream open reading frames are contemplatedby the present invention. Specifically, polypeptides of the inventioncomprise the following amino acid sequences:MVPGAAGWCCLVLWLPACVAAHGFRIHDYLYFQVLSPGDIRYIFTATPAKDFGGIFHTRYEQIHLVPAEPPEACGELSNGFFIQDQIALVERGGCSFLSKTRVVQEHGGRAVIISDNAVDNDSFYVEMIQDSTQRTADIPALFLLGRDGYMIRRSLEQHGLPWAIISIPVNVTSIPTFELLQPPWTFW (SEQ ID NO: 394) andVDNDSFYVEMIQDSTQRTADIPALFLLGRDGYMIRRSLEQHGLPWAIISIPVNV TSIPTFELLQPPWTFW(SEQ ID NO: 395). Polynucleotides encoding these polypeptides are alsoprovided.

[0390] The gene encoding the disclosed cDNA is believed to reside onchromosome 2. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 2.

[0391] This gene is expressed primarily in breast lymph node, ovary,osteoclast cells, and to a lesser extent in human jurkat membrane-boundpolysomes and human placenta.

[0392] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, breastcancer and immune diseases and/or disorders. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., reproductive, endocrine, skeletal,bone, placental, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, amniotic fluid, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0393] The tissue distribution in human breast and placental tissueindicates that the protein product of this gene may be useful fordiagnosis and intervention of tumors within these tissues, in additionto other tumors and tissues where expression has been indicated.Expression in cells of lymphoid origin, the natural gene product may beinvolved in immune functions. Therefore it may be also used as an agentfor immunological disorders including arthritis, asthma, immunedeficiency diseases such as AIDS, and leukemia. Furthermore, the proteinmay also be used to determine biological activity, raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0394] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:60 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1147 of SEQID NO:60, b is an integer of 15 to 1161, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:60, and whereb is greater than or equal to a +14.

[0395] Features of Protein Encoded by Gene No: 51

[0396] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: IATAALFFFFYCQVAGFIGKGQSLRSWVPQRLLGLEPQLQPMQQSRLLLPFLFFLLEGCAPSSLGPGAAPGSGHSLGPPGSPGAPGPQPAVGPSSPCQPGPSPSSPAAAAASSQSSVASWPCTLRCAAPSPDASALRPAASPAATPAWSPGSGTIRVLRPPAPAAAPATAITNRGPPRRRRRNARTA (SEQ ID NO: 396). Polynucleotides encodingthese polypeptides are also provided.

[0397] In yet another embodiment, polypeptides of the invention comprisethe following amino acid sequence:ERPPPRRTGTPVARPRGPPDPAVAAGTALRAKQFARYGAASGVVPGSLWPSPEQLRELEAEEREWYPSLATMQESLRVKQLAEEQKRREREQHIAECMAKMPQMIVNWQQQQRENWEKAQADKERRARLQAEAQELLGYQVDPRSARFQELLQDLEKKERNPQGGKTETEEGGATAALAAAVAQDPAASGAPSS (SEQ ID NO: 397).Polynucleotides encoding these polypeptides are also provided. Thepolypeptide sequence of the latter embodiment was found to have homologyto the human HPK/GCK-like kinase HGK (See Genbank Accession No.gb|AAD16137.1| (AF096300); all references available through thisaccession are hereby incorporated herein by reference; for example, J.Biol. Chem. 274 (4), 2118-2125 (1999)) which is thought to play a rolein modulating gene expression, particularly for genes involved in thec-jun pathway. Based on the sequence similarity, the translation productof this gene is expected to share at least some biological activitieswith signaling and kinase proteins. Such activities are known in theart, some of which are described elsewhere herein. In anotherembodiment, translated products of this gene shares homology withSTE20-related protein kinases (see Genbank Accessions AAD16137 andAAC53165; all references available through this accession are herebyincorporated herein by reference; for example, Yao, Z., et al., J. Biol.Chem. 274 (4), 2118-2125 (1999) and Su, Y. C., et al., EMBO J. 16 (6),1279-1290 (1997)). Based on the sequence similarity, translationproducts of this gene are expected to share at least some biologicalactivities with STE20-related protein kinases. Preferred polypeptidescomprising the amino acid sequences:MQESLRVKQLAEEQKRREREQHIAECMAKMPQMIVNWQQQQRENWEKAQADKERRARLQAEAQELLGYQVDPRSARFQELLQDLEKKERKRLKEEKQKRKKEARAAALAAAVAQDPAASGAPSS (SEQ ID NO: 398) are contemplated by thepresent invention. Polynucleotides encoding these polypeptides are alsoprovided.

[0398] The gene encoding the disclosed cDNA is believed to reside onchromosome 19. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 19.

[0399] This gene is expressed primarily in HL-60, PMA 4H and to a lesserextent in Soares breast 2NbHBst, Human Pituitary, subt IX, and HumanFetal Kidney.

[0400] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immune,hematopoietic, developmental, and proliferative diseases and/ordisorders, particularly promyelocytic leukemia. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., immune, hematopoietic,reproductive, developmental, proliferative, and cancerous and woundedtissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO:182 as residues: Ser-54 to Ser-63, Asn-132 to Thr-145.Polynucleotides encoding said polypeptides are also provided.

[0401] The tissue distribution in HL-60 cells indicates polynucleotidesand polypeptides corresponding to this gene are useful for the diagnosisand treatment of a variety of immune system disorders. Representativeuses are described in the “Immune Activity” and “Infectious Disease”sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, andelsewhere herein. Briefly, the expression of this gene product indicatesa role in regulating the proliferation; survival; differentiation;and/or activation of hematopoietic cell lineages, including blood stemcells. Involvement in the regulation of cytokine production, antigenpresentation, or other processes suggests a usefulness in the treatmentof cancer (e.g. by boosting immune responses). Expression in cells oflymphoid origin, the natural gene product would be involved in immunefunctions. Therefore it is also useful as an agent for immunologicaldisorders including arthritis, asthma, immunodeficiency diseases such asAIDS, leukemia, rheumatoid arthritis, granulomatous disease,inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia,psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity;immune reactions to transplanted organs and tissues, such ashost-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.

[0402] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. Thus, this gene productis thought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0403] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:61 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 673 of SEQID NO:61, b is an integer of 15 to 687, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:61, and where bis greater than or equal to a +14.

[0404] Features of Protein Encoded by Gene No: 52

[0405] The translation product of this gene shares sequence homologywith the human hypothetical L1 protein (third intron of gene TS) (SeeGenbank Accession No. pir|JU0033|JU0033), which is thought to beimportant for the regulation of RNA-dependent DNA polymerases. Preferredpolypeptides comprise the following amino acid sequence:YQSLAETQQKKENFRPISLKNTDAKILNKILANQIQQ KKLIHNDRVGFIPEMQGWFNICKSINIVHHINRTKDKNHMHSIDAEKAFDKIRQSFMLKTLNKLGIHG MYLGR (SEQ ID NO:399), KKENFRPISLKNTDAKILNKILANQIQQHIKKLIHNDRVGFIPEMQGWFNICKSI NIVHNRTKDKNHMIISIDAEKAFDKIRQSFMLKTLNKLGIHGMY (SEQ ID NO: 400), DAKILNKILAN(SEQ ID NO: 401), IQQHIKKLIH (SEQ ID NO: 402), KDKNHMIISIDAEKAFDKI (SEQID NO: 403), MLKTLNKLGI (SEQ ID NO: 404), and/or KKENFRPISL (SEQ ID NO:405). Polynucleotides encoding these polypeptides are also provided.

[0406] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: WTMFIDLHMLNQPCISGMKPTRSLWISFLMCCWIWFANILLRIFASVFFRDIGLKFSFFCCVSARLWYQDDAGLINELGRIPSFY (SEQ ID NO: 406). Polynucleotidesencoding these polypeptides are also provided. The presence of the aminoacid sequences upstream of the predicted signal sequence of the latterembodiment may alter the characteristics of the protein of the presentinvention such that either the full protein, or fragments thereof, arebound to the membrane in a form analogous to a Type II membrane protein.This form of the protein is thought to have a cytoplasmic tail coveringabout the first 21 amino acids. Based on the structural similarity, thetranslation product of this latter embodiment is expected to share atleast some biological activities with type II membrane proteins. Suchactivities are known in the art, some of which are described elsewhereherein.

[0407] This gene is expressed primarily in ulcerative colitis.

[0408] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,gastrointestinal diseases and/or disorders, particularly ulcerativecolitis. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thedigestive system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., gastrointestinal, and cancerous and wounded tissues) or bodilyfluids (e.g., lymph, chyme, bile, serum, plasma, urine, synovial fluidand spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0409] The tissue distribution in ulcerative colon tissue combined withits homology to an RNA-dependent DNA polymerase regulatory protein maysuggest that polynucleotides and polypeptides corresponding to this geneare useful for diagnosis and intervention of tumors and otherproliferative conditions within the indicated tissues, and to a lesserextent in other tissues and cell types.

[0410] Moreover, the expression within cellular sources marked byproliferating cells indicates this protein may play a role in theregulation of cellular division, and may show utility in the diagnosis,treatment, and/or prevention of developmental diseases and disorders,including cancer, and other proliferative conditions. Representativeuses are described in the “Hyperproliferative Disorders” and“Regeneration” sections below and elsewhere herein. Briefly,developmental tissues rely on decisions involving cell differentiationand/or apoptosis in pattern formation. The protein is useful inmodulating the immune response to aberrant polypeptides, as may exist inproliferating and cancerous cells and tissues. The protein can also beused to gain new insight into the regulation of cellular growth andproliferation. Furthermore, the protein may also be used to determinebiological activity, to raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0411] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:62 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 504 of SEQID NO:62, b is an integer of 15 to 518, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:62, and where bis greater than or equal to a +14.

[0412] Features of Protein Encoded by Gene No: 53

[0413] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: ERPEEGTEPSPSPVAEQASVSMTPVFRAWGLWVYVL (SEQ ID NO: 407)PTGFPGPCCMMLLELFPKESVPQAYQGILLYLHFGF.

[0414] Polynucleotides encoding these polypeptides are also provided.

[0415] This gene is expressed primarily in ovary, testis, Hodkin'slymphoma, resting T-Cell; re-excision and to a lesser extent in soaresmultiple sclerosis, human corpus colosum, and fetal kidney.

[0416] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,reproductive, immune, and hematopoietic diseases and/or disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the immunesystem, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g.,reproductive, ovarian, testicular, breast, immune, hematopoietic, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,seminal fluid, breast milk, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0417] The tissue distribution in testicular tissue indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and diagnosis of conditions concerning propertesticular function (e.g. endocrine function, sperm maturation), as wellas cancer. Therefore, this gene product is useful in the treatment ofmale infertility and/or impotence. This gene product is also useful inassays designed to identify binding agents, as such agents (antagonists)are useful as male contraceptive agents. Similarly, the protein isbelieved to be useful in the treatment and/or diagnosis of testicularcancer. The testes are also a site of active gene expression oftranscripts that is expressed, particularly at low levels, in othertissues of the body. Therefore, this gene product may be expressed inother specific tissues or organs where it may play related functionalroles in other processes, such as hematopoiesis, inflammation, boneformation, and kidney function, to name a few possible targetindications. Moreover, the protein product of this gene has also beenshown to be expressed in ovary and breast tissue which, in combinationwith the detected expression in testis, indicates that this proteinrepresents a secreted factor that plays an important role in properreproduction (e.g., hormone, signaling factor, etc.). Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0418] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:63 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 897 of SEQID NO:63, b is an integer of 15 to 911, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:63, and where bis greater than or equal to a +14.

[0419] Features of Protein Encoded by Gene No: 54

[0420] When tested against U937 cell lines, supernatants removed fromcells containing this gene activated the GAS (gamma activating sequence)promoter element. Thus, it is likely that this gene activates myeloidcells, and to a lesser extent, other cells and tissue cell-types,through the JAK-STAT signal transduction pathway. GAS is a promoterelement found upstream of many genes which are involved in the Jak-STATpathway. The Jak-STAT pathway is a large, signal transduction pathwayinvolved in the differentiation and proliferation of cells. Therefore,activation of the Jak-STAT pathway, reflected by the binding of the GASelement, can be used to indicate proteins involved in the proliferationand differentiation of cells.

[0421] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: RGEVPHQPHPTRRTVVSGQAPWXPGPXALGQXVETAAGMGMPLVTVTAATFPTLSCPPRAWPEVEAPEAPALPVVPELPEVPMEMPLVLPPELELLSLEAVHRYQXGGTLMGWTRAEASANGS (SEQ ID NO: 408). Polynucleotides encoding thesepolypeptides are also provided. In yet another embodiment, preferredpolypeptides of the invention comprise the following amino acidsequence: MVLDPYRAVALELQANREPDFSSLVSPLSPRRMAARVFYLLLGECMHVCVCMWGRDTETRGPYRDSPDLPSPRLLTSALSATDSSRETRKAIWSPPDPAGAQIPLRLESIYKAARKPATSSKPRRASLKKKKK (SEQ ID NO: 409). Polynucleotides encodingthese polypeptides are also provided. Polypeptides of the latterembodiment share homology to the human hHR21spB (See Genbank AccessionNo.gi|4101480|gb|AAD01193.1| (AF006264); all references availablethrough this accession are hereby incorporated by reference herein)which is thought to play a role in DNA repair. Based on the sequencesimilarity, the translation product of this gene is expected to share atleast some biological activities with DNA repair proteins. Suchactivities are known in the art, some of which are described elsewhereherein.

[0422] The gene encoding the disclosed cDNA is believed to reside onchromosome 22. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 22.

[0423] This gene is expressed primarily in resting T-Cells, testis,uterine cancer, bone marrow, and to a lesser extent in cerebellum.

[0424] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immune,reproductive, and neural diseases and/or disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., immune, hematopoietic, neural,reproductive, and cancerous and wounded tissues) or bodily fluids (e.g.,lymph, serum, plasma, seminal fluid, amniotic fluid, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0425] The tissue distribution in bone marrow and resting T-cells,combined with the detected GAS biological activity, indicatespolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Briefly, the expression of this geneproduct indicates a role in regulating the proliferation; survival;differentiation; and/or activation of hematopoietic cell lineages,including blood stem cells. Involvement in the regulation of cytokineproduction, antigen presentation, or other processes suggests ausefulness in the treatment of cancer (e.g. by boosting immuneresponses). Expression in cells of lymphoid origin, the natural geneproduct would be involved in immune functions. Therefore it is alsouseful as an agent for immunological disorders including arthritis,asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoidarthritis, granulomatous disease, inflammatory bowel disease, sepsis,acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such asT-cell mediated cytotoxicity; immune reactions to transplanted organsand tissues, such as host-versus-graft and graft-versus-host diseases,or autoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, andscleroderma.

[0426] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. Thus, this gene productis thought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Polynucleotides and polypeptidescorresponding to this gene are useful for the detection, treatment,and/or prevention of neurodegenerative disease states, behavioraldisorders, or inflammatory conditions. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0427] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:64 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 949 of SEQID NO:64, b is an integer of 15 to 963, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:64, and where bis greater than or equal to a +14.

[0428] Features of Protein Encoded by Gene No: 55

[0429] The translation product of this gene was shown to have homologyto the human platelet membrane glycoprotein V, which is a part of theIb-V-1× system of surface glycoproteins (GPs Ib alpha, Ib beta, V, IX)that constitute the receptor for von Willebrand factor (vWf) and mediatethe adhesion of platelets to injured vascular surfaces in the arterialcirculation, a critical initiating event in hemostasis (See GenbankAccession No.gi|388760).

[0430] Moreover, the protein product of this gene was also shown to havehomology to human toll and toll-like receptors (See Genbank AccessionNos. W86352, and gb|AF051151|AF051151; all references available throughthis accession are hereby incorporated herein by reference; for example,Blood 91 (11), 4020-4027 (1998)). Based on the sequence similarity, thetranslation product of this gene is expected to share at least somebiological activities with toll-receptor proteins. Such activities areknown in the art, some of which are described elsewhere herein.Preferred are polypeptides comprising the following amino acid sequence:AFRNLPNLRIL (SEQ ID NO: 410), and/or AFQGLFHLFELRL SEQ ID NO: 411(SEQ IDNo: 399). Polynucleotides encoding these polypeptides are also provided.

[0431] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: NKXILEVPSARTTRIMGDHLDLLLGVVLMAGPVFGIPSCSFDGRIAFYRFCNLTQVPQVLNTTERLLLSFNYIRTVTASSFPFLEQLQLLELGSQYTPLTIDKEAFRNLPNLRILDLGSSKIYFLHPDAFQGLFHLFELRLYFCGLSDAVLKDGYFRNLKALTRLDLSKNQIRSLYLHPSFGKLNSLKSIDFSSNQIFLVCEHELE (SEQ ID NO: 412).Polynucleotides encoding these polypeptides are also provided.

[0432] This gene is expressed primarily in pancreatic tumors.

[0433] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,pancreatic cancer; impaired pancreatic function; altered carbohydratemetabolism; and immune and hematopoietic diseases and/or disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the pancreas orendocrine system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., pancreatic, gastrointestinal, immune, hematopoietic, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, bile, synovial fluid and spinal fluid) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

[0434] The tissue distribution in pancreatic tumors indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and/or treatment of disorders of the pancreas.Expression of this gene product in pancreas tumors indicates a potentialinvolvement in pancreatic cancer, and indicates that the gene productmay play more general roles in cellular proliferation and/or apoptosisas well. Alternately, expression in the pancreas may suggest a generalinvolvement in pancreatic function, and implicate the utility of thisgene product in a variety of pancreatic disorders. Alternately, as thisprotein is a secreted protein, it may simply be produced by the pancreasto have effects at other sites within the body or endocrine system. Inaddition, the homology to a conserved receptor for von Willebrand factorindicates that polynucleotides and polypeptides corresponding to thisgene are useful for the treatment and diagnosis of hematopoetic relateddisorders such as anemia, pancytopenia, leukopenia, thrombocytopenia orleukemia since stromal cells are important in the production of cells ofhematopoietic lineages. The uses include bone marrow cell ex vivoculture, bone marrow transplantation, bone marrow reconstitution,radiotherapy or chemotherapy of neoplasia.

[0435] The gene product may also be involved in lymphopoiesis,therefore, it can be used in immune disorders such as infection,inflammation, allergy, immunodeficiency etc. The product of this genemay also show utility in the treatment of vascular diseases such asathlerosclerosis and stroke. The protein is useful in modulating theimmune response to aberrant polypeptides, as may exist in proliferatingand cancerous cells and tissues. The protein can also be used to gainnew insight into the regulation of cellular growth and proliferation.Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0436] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:65 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 987 of SEQID NO:65, b is an integer of 15 to 1001, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:65, and whereb is greater than or equal to a +14.

[0437] Features of Protein Encoded by Gene No: 56

[0438] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: AHAALQLSLRTCGPCSSPYPHAGLAALLTHMWALQLSLPTCGLAALLTHMRPCSSPYPHAGLAALLTIIMGPCRSPYPHGGLAAVLTHMRALQLSLPTWGLAALLTHMRPCSSPYPHAGLACCWLWSLSSHRSLQVQATHRLVVRTIKDRVMLKVLPQTRRRGPFLSSCRNDVMRNCVPRHAVLVTTCVFVSFPTHCKVGITGPITQVKQKPGNHSSPCPVIQLVAKAEFELMLPSVPKPVYLTLVLSCWCLCDVPCLSVS L (SEQ ID NO:413). Polynucleotides encoding these polypeptides are also provided. Ithas been determined that the protein product of this gene has aconserved G-protein receptor motif beginning at amino acid position 89and ending at amino acid position 105 of the amino acid sequencereferenced in Table 1 for this gene.

[0439] Preferred polypeptides of the invention comprise the followingamino acid sequence: LACCWLWSLSSHRSLQV (SEQ ID NO: 414). Polynucleotidesencoding these polypeptides are also provided.

[0440] This gene is expressed primarily in tonsils and anergic T-cells.

[0441] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immunesystem disorders; immune dysfunction; impaired immune surveillance.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the immunesystem, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g.,immune, hematopoietic, and cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder. Preferred polypeptides of thepresent invention comprise immunogenic epitopes shown in SEQ ID NO:187as residues: Pro-22 to Pro-28, Pro-41 to His-48, Pro-79 to His-86,Pro-126 to Phe-134, Ser-137 to Met-143, Gln-176 to Ser-186.Polynucleotides encoding said polypeptides are also provided.

[0442] The tissue distribution in T-cells and tonsils, combined with theidentification of a G-protein receptor motif within the open readingframe, indicates polynucleotides and polypeptides corresponding to thisgene are useful for the diagnosis and treatment of a variety of immunesystem disorders. Representative uses are described in the “ImmuneActivity” and “Infectious Disease” sections below, in Example 11, 13,14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, theexpression of this gene product indicates a role in regulating theproliferation; survival; differentiation; and/or activation ofhematopoietic cell lineages, including blood stem cells. Involvement inthe regulation of cytokine production, antigen presentation, or otherprocesses suggests a usefulness in the treatment of cancer (e.g. byboosting immune responses). Expression in cells of lymphoid origin, thenatural gene product would be involved in immune functions. Therefore itis also useful as an agent for immunological disorders includingarthritis, asthma, immunodeficiency diseases such as AIDS, leukemia,rheumatoid arthritis, granulomatous disease, inflammatory bowel disease,sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities,such as T-cell mediated cytotoxicity; immune reactions to transplantedorgans and tissues, such as host-versus-graft and graft-versus-hostdiseases, or autoimmunity disorders, such as autoimmune infertility,lense tissue injury, demyelination, systemic lupus erythematosis, druginduced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, andscleroderma.

[0443] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. Thus, this gene productis thought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0444] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:66 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1544 of SEQID NO:66, b is an integer of 15 to 1558, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:66, and whereb is greater than or equal to a +14.

[0445] Features of Protein Encoded by Gene No: 57

[0446] A translated product of this gene shares homology with Krueppelfamily zinc finger proteins (see Genbank Accession AAB86596; allreferences available through this accession are hereby incorporatedherein by reference, for example, Hussey, D. J., et al., Genomics 45(2), 451-455 (1997)). Based on the sequence similarity, a translationproduct of this gene is expected to share at least some biologicalactivities with zinc finger proteins. Such activities are known in theart, some of which are described elsewhere herein.

[0447] Preferred polypeptides of the invention comprise the amino acidsequence EIGSHSVAQAGLELPGSSDPPTSGSQSAGITGVSQGTQPSVDLCQEEPAGADQP HGSLQ(SEQ ID NO: 415). Polynucleotides encoding this polypeptide are alsoprovided.

[0448] This gene is expressed primarily in healing groin wound (6.5hours post incision), and to a lesser extent in testis.

[0449] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, woundedtissues; disorders involving tissue repair; male reproductive disorders;mucositis; tissue degeneration. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe reproductive system, expression of this gene at significantly higheror lower levels may be routinely detected in certain tissues or celltypes (e.g., reproductive, testis, and cancerous and wounded tissues) orbodily fluids (e.g., lymph, serum, plasma, urine, seminal fluid,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO: 188 as residues: Ser-59 to Gly-68. Polynucleotides encoding saidpolypeptides are also provided.

[0450] The tissue distribution in healing groin wound and testisindicates that polynucleotides and polypeptides corresponding to thisgene are useful for therapeutic use as an agent to facilitate woundhealing and tissue regeneration. Expression of this product during woundhealing indicates that it may play a beneficial role during the process.Alternately, expression during wound healing may also suggest that itplays a negative role during the process, e.g. fibrosis and scarring,and that therapeutics designed to counter the effects of this proteinmay be even more beneficial. In addition, expression of this proteinwithin the groin and testis indicates that it may play a role inreproductive system function—particularly male reproductive function—andthat this protein may even have potential uses as a male contraceptive.Alternately, the tissue distribution in testicular tissue indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and diagnosis of conditions concerning propertesticular function (e.g. endocrine function, sperm maturation), as wellas cancer. Therefore, this gene product is useful in the treatment ofmale infertility and/or impotence. This gene product is also useful inassays designed to identify binding agents, as such agents (antagonists)are useful as male contraceptive agents. Similarly, the protein isbelieved to be useful in the treatment and/or diagnosis of testicularcancer. The testes are also a site of active gene expression oftranscripts that is expressed, particularly at low levels, in othertissues of the body. Therefore, this gene product may be expressed inother specific tissues or organs where it may play related functionalroles in other processes, such as hematopoiesis, inflammation, boneformation, and kidney function, to name a few possible targetindications. Furthermore, the protein may also be used to determinebiological activity, to raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0451] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:67 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1308 of SEQID NO:67, b is an integer of 15 to 1322, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:67, and whereb is greater than or equal to a +14.

[0452] Features of Protein Encoded by Gene No: 58

[0453] A preferred polypeptide fragment of the invention comprises thefollowing amino acid sequence:MGEASPPAPARRHLLVLLLLLSTLVPSAAAPIHDADAQESSLGLTGLQSLLQG FSRLFLKVTCFGA (SEQID NO: 416). Polynucleotides encoding these polypeptides are alsoprovided.

[0454] This gene is expressed primarily in testis, and to a lesserextent in brain and fetal heart.

[0455] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,neurodegenerative disorders; psychological disorders; learningdisabilities; altered heart function; altered male reproductivefunction. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thebrain and nervous system, cardiovascular system, or reproductive system,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types (e.g., reproductive,testis, developmental, and cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, seminal fluid, synovial fluidand spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO:189 as residues: Pro-82 to His-93. Polynucleotides encoding saidpolypeptides are also provided.

[0456] The tissue distribution in testicular tissue indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and diagnosis of conditions concerning propertesticular function (e.g. endocrine function, sperm maturation), as wellas cancer. Therefore, this gene product is useful in the treatment ofmale infertility and/or impotence. This gene product is also useful inassays designed to identify binding agents, as such agents (antagonists)are useful as male contraceptive agents. Similarly, the protein isbelieved to be useful in the treatment and/or diagnosis of testicularcancer. The testes are also a site of active gene expression oftranscripts that is expressed, particularly at low levels, in othertissues of the body. Therefore, this gene product may be expressed inother specific tissues or organs where it may play related functionalroles in other processes, such as hematopoiesis, inflammation, boneformation, and kidney function, to name a few possible targetindications. Alternatively, the tissue distribution in brain indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for the diagnosis and/or treatment of brain and nervous systemdisorders. Expression of this gene product in a variety of brain regionsindicates a role in brain and nervous system function. This indicatesthat the protein product may be useful in the treatment ofneurodegenerative disorders; learning disabilities; psychoses; andbehaviors, including feeding; sleeping; perception; balance; etc.Therefore, this gene product may be useful in the treatment of a varietyof heart conditions, including myocardial infarction; congestive heartfailure; arrhythmias; coronary occlusion; and a variety of otherdisorders of the heart. The secreted protein can also be used todetermine biological activity, to raise antibodies, as tissue markers,to isolate cognate ligands or receptors, to identify agents thatmodulate their interactions, and as nutritional supplements. It may alsohave a very wide range of biological activities. Representative uses aredescribed in the “Chemotaxis” and “Binding Activity” sections below, inExamples 11, 12, 13, 14, 15, 16, 18, 19, and 20, and elsewhere herein.Briefly, the protein may possess the following activities: cytokine,cell proliferation/differentiation modulating activity or induction ofother cytokines; immunostimulating/immunosuppressant activities (e.g.for treating human immunodeficiency virus infection, cancer, autoimmunediseases and allergy); regulation of hematopoiesis (e.g. for treatinganemia or as adjunct to chemotherapy); stimulation or growth of bone,cartilage, tendons, ligaments and/or nerves (e.g. for treating wounds,stimulation of follicle stimulating hormone (for control of fertility);chemotactic and chemokinetic activities (e.g. for treating infections,tumors); hemostatic or thrombolytic activity (e.g. for treatinghemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g.for treating septic shock, Crohn's disease); as antimicrobials; fortreating psoriasis or other hyperproliferative diseases; for regulationof metabolism, and behavior. Also contemplated is the use of thecorresponding nucleic acid in gene therapy procedures. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0457] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:68 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 851 of SEQID NO:68, b is an integer of 15 to 865, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:68, and where bis greater than or equal to a +14.

[0458] Features of Protein Encoded by Gene No: 59

[0459] The translation product of this gene shares sequence homologywith alpha 1,3 galactosyltransferase which is thought to be important inthe regulation of protein glycosylation and sugar transfer (See GenbankAccession No. bs|150271; all references available through this accessionare hereby incorporated by reference herein). Preferred polypeptidescomprise the following amino acid sequence:MLVVSTVIIVFWEFINSTEGSFLWIYHSKNPEVDDSSAQKGWWFLSWFNNGIHNYQQGEEDIDKEKGREETKGRKMTQQSFGYGTGLIQT (SEQ ID NO: 417), and/orFPGRTHASGNVKGKVILS (SEQ ID NO: 418). Polynucleotides encoding thesepolypeptides are also provided.

[0460] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: ADQEKIRNVKGKVILSMLVVSTVIIVFWEFINSTEGSFLWIYHSKNPEVDDSSA (SEQ IDNO: 419) QKGWWFLSWFNNGIHN YQQGEEDIDKEKGREETKGRKMTQQSFGYGTGLIQT.

[0461] Polynucleotides encoding these polypeptides are also provided.The presence of the upstream amino acids of the latter embodiment maysignificantly alter the secreted characteristics of the presentinvention. Namely, either the full-length protein, or fragments thereof,may become membrane bound in a mechanism analogous to type II membraneproteins. Based on the such characteristics, the translation product ofthis latter embodiment is expected to share at least some biologicalactivities with type II membrane proteins. Such activities are known inthe art, some of which are described elsewhere herein fragments.

[0462] The gene encoding the disclosed cDNA is believed to reside onchromosome 9. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 9.

[0463] This gene is expressed primarily in primary dendritic cells,neutrophils, and T cells and to a lesser extent in liver hepatoma andinfant brain.

[0464] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immunedysfunction, hematopoietic disorders; inflammation; neurodegenerativedisorders; liver hepatoma; T cell lymphoma. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, liver, or CNS, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., immune, hematopoietic, neural,and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 190 as residues: His-27 toGly-41, Gln-56 to Tyr-83. Polynucleotides encoding said polypeptides arealso provided.

[0465] The tissue distribution in dendritic cells, combined with thehomology to galactosyltransferases indicates that polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosisand/or treatment of a variety of disorders, particularly of the immuneand nervous systems since normal function of such tissues depends uponproper glycoprotein recognition and galactosyltransferase function.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Expression of this gene product indendritic cells indicates a role in the regulation of the immune systemand responses to infectious agents. This may involve roles in antigenpresentation, antigen processing, stimulation and activation of B and Tcells, or stimulation/activation of dendritic cells themselves. This maybe evidenced by effects on cytokine production. Expression of this geneproduct in other hematopoietic cells such as T cells and neutrophilsalso indicates roles in the functions of those cells as well, andinvolvement in the proliferation, survival, and/or differentiation ofhematopoietic cells in general. In addition, the expression alsoindicates that polynucleotides and polypeptides corresponding to thisgene are useful for the treatment and diagnosis of hematopoetic relateddisorders such as anemia, pancytopenia, leukopenia, thrombocytopenia orleukemia since stromal cells are important in the production of cells ofhematopoietic lineages. The uses may include bone marrow cell ex vivoculture, bone marrow transplantation, bone marrow reconstitution,radiotherapy or chemotherapy of neoplasia.

[0466] The gene product may also be involved in lymphopoiesis,therefore, it can be used in immune disorders such as infection,inflammation, allergy, immunodeficiency etc. Expression of this geneproduct within infant brain also indicates a role in neuron survival,synapse formation, neurotransmission, perception, etc. The protein isuseful in the treatment and/or prevention of degenerative myelinatingdiseases and/or disorders, particularly multiple sclerosis, in additionto other disorders which occur secondary to aberrant fatty-acidmetabolism. Furthermore, the protein may also be used to determinebiological activity, raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0467] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:69 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1136 of SEQID NO:69, b is an integer of 15 to 1150, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:69, and whereb is greater than or equal to a +14.

[0468] Features of Protein Encoded by Gene No: 60

[0469] The translation product of this gene shares homology withserine/threonine kinases (see Genbank Accessions AAA36658 and AAB97983;all references available through these accessions are herebyincorporated herein by reference, for example Levedakou, E. N., et al.,Oncogene 9, 1977-1988 (1994)).

[0470] This gene is expressed primarily in small intestine andleukocytes.

[0471] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,hematopoietic disorders; inflammation; allergy; impaired immunity;autoimmunity, and gastrointestinal disorders. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., gastrointestinal, immune,hematopoietic, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder.

[0472] The tissue distribution in leukocytes indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and/or treatment of a variety of hematopoieticdisorders. Representative uses are described in the “Immune Activity”and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18,19, 20, and 27, and elsewhere herein. Expression of this gene product insmall intestines and leukocytes indicates that it may be expressed byvarious hematopoietic cells, for example, in the peyer's patches ofintestine as well as within the circulation itself. Thus, it may play arole in the proliferation; survival; differentiation; or activation ofvarious hematopoietic cell lineages. This may affect the cells' abilityto recognize antigen; mount an immune response; participate ininflammatory processes; and effectively patrol the body for infectiousor foreign agents. Alternately, expression of this gene product in smallintestine may reflect a role in digestion and food processing.Furthermore, the protein may also be used to determine biologicalactivity, raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0473] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:70 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1384 of SEQID NO:70, b is an integer of 15 to 1398, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:70, and whereb is greater than or equal to a +14.

[0474] Features of Protein Encoded by Gene No: 61

[0475] The translation product of this gene shares sequence homologywith the Drosophila strabismus gene product which is thought to regulatetissue polarity and cell fate decisions (See Genbank AccessionNo.gi|2854044 (AF044208); all references available through thisreference are hereby incorporated herein by reference).

[0476] When tested against U937, SK, Raji, and Reh cell lines,supernatants removed from cells containing this gene activated the GAS(gamma activating sequence) promoter element. Thus, it is likely thatthis gene activates myeloid cells, and to a lesser extent, other cellsand tissue cell types, through the JAK-STAT signal transduction pathway.GAS is a promoter element found upstream of many genes which areinvolved in the Jak-STAT pathway. The Jak-STAT pathway is a large,signal transduction pathway involved in the differentiation andproliferation of cells. Therefore, activation of the Jak-STAT pathway,reflected by the binding of the GAS element, can be used to indicateproteins involved in the proliferation and differentiation of cells.

[0477] Preferred polypeptides of the invention comprise the followingamino acid sequence:MQSPLVECPPPSIHYWPSVPAGAQGACSPMFHAAGWSRSQPNGEIPASSXGH (SEQ ID NO: 420)LSIQRAALVVLENYYKDFTIYNPNLLTASKFRAAKHMAGLKVYNVDGPSNNATGQSRAMIAAAARRRDSSHNELYYEEAEHERRVKKRKARLVVAVEEAFIHIQRLQAEEQQKAPGEVMDPREAAQAIFPSMARALQKYLRITRQQNYHSMESILQAPGLLHHQRHDPQGLPRTVPQCGPHPAI, LSIQRAALVVLENYYKDFTIYNP, (SEQ ID NO: 421)DSSHNELYYEEAEHE, and/or (SEQ ID NO: 422) FPSMARALQKYLRITRQQ. (SEQ ID NO:423)

[0478] Polynucleotides encoding these polypeptides are also provided. Apreferred polypeptide fragment of the invention comprises the followingamino acid sequence:MAFKLLILLIGTWALFFRKRRADMPRVFVFRALLLVLIFLFCGFPIGFFTGSAF (SEQ ID NO: 424)WTLGNRNYQGIVQYAVSPCGMPSSFHPLLAIRPCWSSGSLQPNVPRCRLVPLPTEWGNPRFQXGTPEYPASSIGGPRKLLQRFHHL.

[0479] Polynucleotides encoding these polypeptides are also provided.

[0480] The translation product of this gene was determined to have atransmembrane domain located at amino acid position 249-266 of the aminosequence referenced in Table 1 for this gene. Likewise, this protein isthought to be a Type II membrane protein. In another embodiment,preferred polypeptides of the present invention comprise the amino acidsequences: MQSPLWMPSSSSITWPSSCWSSGSCSPCSRCRWSRSTDGESRFYSLGHL, (SEQ IDNO: 425) MQSPLWMPSSSSITWPSSCWSSGSCSPCSRCRWSRSTDGESRFYSLGHLSIQR (SEQ IDNO: 426) AALVVLENYYKDFTIYNPNLLTASKFRAAKHMAGLKVYNVDGPSNNATGQSRAMIAAAARRRDSSHNELYYEEAEHERRVKKRKARLVVAVEEAFIHIQRLQAEEQQKAPGEVMDPREAAQAIFPSMARALQKYLRITRQQNYHSMESILQHLAFCITNGMTPKAFLERYLSAGPTLQYDKDRWLSTQWRLVSDEALTNGLRDGIVFVLKCLDFSLVVNVKKIPFIILSEEFIDPKSHKFVLRLQSETSV,MAFKLLILLIGTWALFFRKRRADMPRVFVFRALLLVLIFLFVVSYWLFYGVRI (SEQ ID NO: 428)LDSRDRNYQGIVQYAVSLVDALLFIHYLAIVLLELRQLQPMFTLQVVRSTDGE SRFYSLGH, andMPRVFVFRALLLVLIFLFVVSYWLFYGVRILDSRDRNYQGIVQYAVSLVDALL (SEQ ID NO: 427)FIHYLAIVLLELRQLQPMFTLQVVRSTDGESRFYSLGHL.

[0481] Polynucleotides encoding these polypeptides are also provided.

[0482] This gene is expressed primarily in human osteoclast stromalcells, fetal liver and spleen, and in endometrial tumors and to a lesserextent in hematopoietic cells, including T-cells and CD34 positive cellsisolated from cord blood, as well as the thymus, fetal heart, 8 week oldwhole embryos, and tumors of pancreatic and testicular origin.

[0483] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immunesystem disorders, including AIDS and other hematopoietic diseases and/ordisorders, in addition to tumors of osteoclast, endometrial, pancreatic,or testicular origin. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune system as well as biological processes involved in cellularproliferation and/or differentiation, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., immune, haematopoeitic, skeletal,cancerous, and/or other tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid, lymph, breast milk,and/or seminal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO:192 as residues: Pro-17 to Gln-24, Asp-86 to Ser-96, Arg-106 toAsn-112, Ala-119 to Ala-130, Ala-148 to Pro-155, Gln-223 to Leu-230.Polynucleotides encoding said polypeptides are also provided.

[0484] The tissue distribution in immune cells and tissues, combinedwith the detected GAS biological activity, indicates polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosis andtreatment of a variety of immune system disorders. Representative usesare described in the “Immune Activity” and “Infectious Disease” sectionsbelow, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhereherein. Briefly, the expression of this gene product indicates a role inregulating the proliferation; survival; differentiation; and/oractivation of hematopoietic cell lineages, including blood stem cells.Involvement in the regulation of cytokine production, antigenpresentation, or other processes suggests a usefulness in the treatmentof cancer (e.g. by boosting immune responses). Expression in cells oflymphoid origin, the natural gene product would be involved in immunefunctions. Therefore it is also useful as an agent for immunologicaldisorders including arthritis, asthma, immunodeficiency diseases such asAIDS, leukemia, rheumatoid arthritis, granulomatous disease,inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia,psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity;immune reactions to transplanted organs and tissues, such ashost-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.

[0485] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. Thus, this gene productis thought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Alternatively, the tissueexpression in liver tissues indicates that polynucleotides andpolypeptides corresponding to this gene are useful for the detection andtreatment of liver disorders and cancers (e.g. hepatoblastoma, jaundice,hepatitis, liver metabolic diseases and conditions that are attributableto the differentiation of hepatocyte progenitor cells). In addition theexpression in fetus would suggest a useful role for the protein productin developmental abnormalities, fetal deficiencies, pre-natal disordersand various would-healing models and/or tissue traumas. Furthermore, theprotein may also be used to determine biological activity, raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0486] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:71 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1543 of SEQID NO:71, b is an integer of 15 to 1557, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:71, and whereb is greater than or equal to a +14.

[0487] Features of Protein Encoded by Gene No: 62

[0488] A preferred polypeptide fragment of the invention comprises thefollowing amino acid sequence: MGLPVSWAPPALWVLGCCALLLSLWALCTACRSPRTL(SEQ ID NO: 429). Polynucleotides encoding these polypeptides are alsoprovided.

[0489] This gene is expressed primarily in human thymus, human synovialsarcoma, and to a lesser extent in breast cancer cells.

[0490] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immunediseases and/or disorders, particularly autoimmune disorders such asarthritis. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,immune, hematopoietic, and cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder. Preferred polypeptides of thepresent invention comprise immunogenic epitopes shown in SEQ ID NO: 193as residues: Pro-40 to Arg-50, Ser-72 to Arg-77, His-82 to Leu-91,Gln-171 to Glu-189, Val-203 to Gly-222, Pro-263 to Thr-269, Ser-282 toTrp-287. Polynucleotides encoding said polypeptides are also provided.

[0491] The tissue distribution in thymus indicates polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosis andtreatment of a variety of immune system disorders. Representative usesare described in the “Immune Activity” and “Infectious Disease” sectionsbelow, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhereherein. Briefly, the expression of this gene product indicates a role inregulating the proliferation; survival; differentiation; and/oractivation of hematopoietic cell lineages, including blood stem cells.Involvement in the regulation of cytokine production, antigenpresentation, or other processes suggests a usefulness in the treatmentof cancer (e.g. by boosting immune responses). Expression in cells oflymphoid origin, the natural gene product would be involved in immunefunctions. Therefore it is also useful as an agent for immunologicaldisorders including arthritis, asthma, immunodeficiency diseases such asAIDS, leukemia, rheumatoid arthritis, granulomatous disease,inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia,psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity;immune reactions to transplanted organs and tissues, such ashost-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.

[0492] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. Thus, this gene productis thought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. The protein is useful in modulatingthe immune response to aberrant polypeptides, as may exist in cancerousand/or proliferative cells and tissues. Furthermore, the protein mayalso be used to determine biological activity, raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0493] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:72 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1149 of SEQID NO:72, b is an integer of 15 to 1163, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:72, and whereb is greater than or equal to a +14.

[0494] Features of Protein Encoded by Gene No: 63

[0495] The translation product of this gene shares sequence homologywith human, porcine, and mouse zona pellucida binding protein sp 38which is known to be important in sperm binding to the zona pellucida ofan egg cell. Monoclonal antibodies directed against this protein haveresulted in inhibition of the sperm/egg binding reaction. As such thetranslation product of this gene may show commercial utility as acontraceptive. (See Genbank Accession No. gnl|PIDId|1005021; allreferences available through this accession are hereby incorporated byreference herein).

[0496] Preferred polypeptides of the invention comprise the followingamino acid sequence: IYGKTGQPDKIYVELHQNSP (SEQ ID NO: 430),FLEPLSGLYTCTLSYK (SEQ ID NO: 431), LQVVRLDSCRPGFGKN (SEQ ID NO: 432),and/or CVSVLTYGAKSC (SEQ ID NO: 433). Polynucleotides encoding thesepolypeptides are also provided.

[0497] This gene is expressed primarily in a human testes library. Ithas not been found in other libraries screened at HGS.

[0498] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,infertility, and/or other reproductive diseases and/or disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the male andfemale reproductive systems, expression of this gene at significantlyhigher or lower levels may be routinely detected in certain tissues orcell types (e.g., testes, and cancerous and wounded tissues) or bodilyfluids (e.g. seminal fluid, lymph, serum, plasma, urine, synovial fluidand spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO: 194 as residues: Lys-35 to Asp-40, Pro-75 to Asn-84, Lys-114 toArg-129, Arg-138 to Ser-143, Ser-154 to Asn-160, Val-224 to Asn-231,Arg-238 to Asp-243, Asp-276 to Asn-291, Lys-324 to Asp-338.Polynucleotides encoding said polypeptides are also provided.

[0499] The tissue distribution in testes combined with the homology tothe human, porcine, and mouse zona pellucida protein Sp 38 indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for the production of a contraceptive vaccine. Alternatively, theprotein may show utility in the diagnosis, treatment, and/or preventionof a variety of reproductive disorders within both the male and femalereproductive systems. This gene product is also useful in assaysdesigned to identify binding agents, as such agents (antagonists) areuseful as male contraceptive agents. Similarly, the protein is believedto be useful in the treatment and/or diagnosis of testicular cancer. Thetestes are also a site of active gene expression of transcripts that isexpressed, particularly at low levels, in other tissues of the body.Therefore, this gene product may be expressed in other specific tissuesor organs where it may play related functional roles in other processes,such as hematopoiesis, inflammation, bone formation, and kidneyfunction, to name a few possible target indications. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0500] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:73 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1472 of SEQID NO:73, b is an integer of 15 to 1486, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:73, and whereb is greater than or equal to a +14.

[0501] Features of Protein Encoded by Gene No: 64

[0502] When tested against U937 cell lines, supernatants removed fromcells containing this gene activated the GAS (gamma activating sequence)promoter element. Thus, it is likely that this gene activates myeloid,and to a lesser extent, other cells and tissue cell types, through theJAK-STAT signal transduction pathway. GAS is a promoter element foundupstream of many genes which are involved in the Jak-STAT pathway. TheJak-STAT pathway is a large, signal transduction pathway involved in thedifferentiation and proliferation of cells. Therefore, activation of theJak-STAT pathway, reflected by the binding of the GAS element, can beused to indicate proteins involved in the proliferation anddifferentiation of cells. Translated products of this gene sharehomology with a human B-cell growth factor molecule (see GenbankAccession AAB02649; all references available through this accession arehereby incorporated herein by reference; for example Sharma S., et al.,Science 235 (4795), 1489-1492 (1987)). Based on sequence similarity,translation products of this gene are expected to share at least somebiological activities with growth factor molecules, more preferably withB-cell growth factor molecules.

[0503] Preferred polypeptides of the invention comprise amino acidsequences: KNNWWQGVVVLACNPSTLGDRGSWIT (SEQ ID NO: 434),SCLGLPKCWDYRQEPPHPATSYFL (SEQ ID NO: 436), and GQEFETRLTNIVKLRLY (SEQ IDNO: 435). Polynucleotides encoding these polypeptides are also provided.

[0504] This gene is expressed primarily an apoptotic T-cell library, andto a lesser extent, in whole embryo.

[0505] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immune,hematopoietic, and developmental diseases and/or disorders, particularlydisorders related to aberrant cell death regulation. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., immune, hematopoietic,developmental, reproductive, apoptotic cells, and cancerous and healingtissue or cells) or bodily fluids (e.g., serum, lymph, amniotic fluid,plasma, urine, synovial fluid and spinal fluid, and/or lymph) or anothertissue or cell sample taken from an individual having such a disorder,relative to the standard gene expression level, i.e., the expressionlevel in healthy tissue or bodily fluid from an individual not havingthe disorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 195 as residues: Met-1 toAla-6, Gly-51 to Gly-71. Polynucleotides encoding said polypeptides arealso provided.

[0506] The tissue distribution in apoptotic T-cells indicatespolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Briefly, the expression of this geneproduct indicates a role in regulating the proliferation; survival;differentiation; and/or activation of hematopoietic cell lineages,including blood stem cells. Involvement in the regulation of cytokineproduction, antigen presentation, or other processes suggests ausefulness in the treatment of cancer (e.g. by boosting immuneresponses). Expression in cells of lymphoid origin, the natural geneproduct would be involved in immune functions. Therefore it is alsouseful as an agent for immunological disorders including arthritis,asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoidarthritis, granulomatous disease, inflammatory bowel disease, sepsis,acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such asT-cell mediated cytotoxicity; immune reactions to transplanted organsand tissues, such as host-versus-graft and graft-versus-host diseases,or autoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, andscleroderma.

[0507] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. Thus, this gene productis thought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. The protein can also be used togain new insight into the regulation of cellular growth andproliferation. Furthermore, the protein may also be used to determinebiological activity, raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0508] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:74 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1539 of SEQID NO:74, b is an integer of 15 to 1553, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:74, and whereb is greater than or equal to a +14.

[0509] Features of Protein Encoded by Gene No: 65

[0510] The translation product of this gene shares sequence homologywith a 50 kDa glycoprotein of the human erythrocyte membrane associatedblood-group antigen which is thought to have a transport or channelfunction in the erythrocyte membrane (See, e.g., Genbank AccessionsHSEPMG50, AAC04247, AAD54392, and CAA45883; all references availablethrough this accession are hereby incorporated herein by reference, forexample Huang, C. H., J. Biol. Chem. 273 (4), 2207-2213 (1998)).

[0511] When tested against Jurkat cell lines, supernatants removed fromcells containing this gene activated the GAS (gamma activating sequence)promoter element. Thus, it is likely that this gene activates T-cells,and to a lesser extent, other cells and tissue cell types, through theJAK-STAT signal transduction pathway. GAS is a promoter element foundupstream of many genes which are involved in the Jak-STAT pathway. TheJak-STAT pathway is a large, signal transduction pathway involved in thedifferentiation and proliferation of cells. Therefore, activation of theJak-STAT pathway, reflected by the binding of the GAS element, can beused to indicate proteins involved in the proliferation anddifferentiation of cells.

[0512] The translation product of this gene has been determined tocontain two transmembrane domains located at amino acid positions95-124, and 1-27 of the amino acid sequence referenced in Table 1 forthis gene. Therefore, this protein may share structural characteristicsto Type IIIa membrane protein. Based on the sequence similarity to thehuman erythrocyte membrane associated blood-group antigen, and thestructural similarity to type IIIa membrane proteins, the translationproduct of this gene is expected to share at least some biologicalactivities with such proteins. Such activities are known in the art,some of which are described elsewhere herein.

[0513] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequences: PAKGEGCRRLHDHPHIWRLLWAHSDPDPLPTQPRAEQGETEFCVPVGPLCHDWHPLPVDVLAQLQLSHILPWGQPAPSRHQHLLLLGSLRAYLGGNIQCPAKKGKLDMVHIQNATLAGGVAVGTAAEMMLMPYGALIIGFVCGIISTLGFVYLTPFLESRLHIQDTCGINNLHGIPGIIGGIVGAVTAASASLEVYGKEGLVHSFDFQGFNGDWTARTQGKFQIYGLLVTLAMALMGGIIVGLILRLPFWGQPSDENCFEDAVYWEMPEGNSTVYIPEDPTFKPSGPSVPSVPMVSPLPMAS SVPLVP (SEQ ID NO: 437) andMTFFQVTLFAVNEFILLNLLKVKDAGGSMTIHTFGAYFGLTVTRILYRRNLEQSKERQNSVYQSDLFAMIGTLFLWMYWPSFNSAISYHGDSQHRAAINTYCSLAACVLTSVAISSALHKKGKLDMVHIQNATLAGGVAVGTAAE (SEQ ID NO: 438).Polynucleotides encoding these polypeptides are also provided.

[0514] The gene encoding the disclosed cDNA is believed to reside onchromosome 18. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 18.

[0515] This gene is expressed primarily in tonsils and to a lesserextent in the larynx, kidney medulla, epithelial cells, keratinocytes,and cells involved in hematopoiesis, especially neutrophils.

[0516] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,hematopoietic diseases and/or disorders, in addition to, theproliferation and/or differentiation of integumentary cells. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., haematopoetic, integumentary,and cancerous and wounded tissues) or bodily fluids (e.g., serum,plasma, urine, synovial fluid and spinal fluid, lymph) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO:196 as residues: Gly-85 toLys-94, Gln-125 to Cys-131, Glu-151 to Gly-159. Polynucleotides encodingsaid polypeptides are also provided.

[0517] The tissue distribution in tonsils, combined with the homology toa 50 kDa glycoprotein of the human erythrocyte membrane proteinindicates polynucleotides and polypeptides corresponding to this geneare useful for the treatment and diagnosis of hematopoietic relateddisorders such as anemia, pancytopenia, leukopenia, thrombocytopenia orleukemia since stromal cells are important in the production of cells ofhematopoietic lineages. Representative uses are described in the “ImmuneActivity” and “Infectious Disease” sections below, in Example 11, 13,14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the usesinclude bone marrow cell ex-vivo culture, bone marrow transplantation,bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.

[0518] The gene product may also be involved in lymphopoiesis,therefore, it can be used in immune disorders such as infection,inflammation, allergy, immunodeficiency etc. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Furthermore,the protein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0519] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:75 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1636 of SEQID NO:75, b is an integer of 15 to 1650, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:75, and whereb is greater than or equal to a +14.

[0520] Features of Protein Encoded by Gene No: 66

[0521] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: PRVRTRAPVVPPAGHRALSPAGVLLAVPAMLSLDFLDDVRRMNKRQVSLSVLFFSWLFLSLRGCCCGARRTPGFWCEGLSWSDTRVIRFLWRLWPEAALSASL FLTPN (SEQ ID NO:439). Polynucleotides encoding these polypeptides are also provided.

[0522] This gene is expressed primarily in hematopoietic tissues,especially helper T-cells and anergic T-cells.

[0523] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,tuberculosis, AIDS, and other immune diseases and/or disorders,particularly infections and/or malignancies. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., haematopoeitic, immune, andcancerous, and/or wounded tissues) or bodily fluids (e.g., serum,plasma, urine, synovial fluid and spinal fluid, and/or lymph) or anothertissue or cell sample taken from an individual having such a disorder,relative to the standard gene expression level, i.e., the expressionlevel in healthy tissue or bodily fluid from an individual not havingthe disorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 197 as residues: Asp-9 toGln-17. Polynucleotides encoding said polypeptides are also provided.

[0524] The tissue distribution in immune cells and tissues indicatespolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Briefly, the expression of this geneproduct indicates a role in regulating the proliferation; survival;differentiation; and/or activation of hematopoietic cell lineages,including blood stem cells. Involvement in the regulation of cytokineproduction, antigen presentation, or other processes suggests ausefulness in the treatment of cancer (e.g. by boosting immuneresponses). Expression in cells of lymphoid origin, the natural geneproduct would be involved in immune functions. Therefore it is alsouseful as an agent for immunological disorders including arthritis,asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoidarthritis, granulomatous disease, inflammatory bowel disease, sepsis,acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such asT-cell mediated cytotoxicity; immune reactions to transplanted organsand tissues, such as host-versus-graft and graft-versus-host diseases,or autoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, andscleroderma.

[0525] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. Thus, this gene productis thought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0526] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:76 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 2136 of SEQID NO:76, b is an integer of 15 to 2150, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:76, and whereb is greater than or equal to a +14.

[0527] Features of Protein Encoded by Gene No: 67

[0528] The polypeptide of this gene has been determined to have atransmembrane domain at about amino acid position 15-34 of the aminoacid sequence referenced in Table 1 for this gene. Moreover, acytoplasmic tail encompassing amino acids 1-14 of this protein has alsobeen determined. Based upon these characteristics, it is believed thatthe protein product of this gene shares structural features to type IImembrane proteins. Translated products of this gene share homology withpolypeptides encoded by human herpes and papilloma viruses (see GenbankAccessions CAA58337 and CAA46991; all references available through theseaccessions are hereby incorporated herein by reference; for example,Gompels, U. A., et al., Virology 209 (1), 29-51 (1995) and Kahn, T., etal., Mol. Carcinog. 6 (2), 88-99 (1992)). Preferred polypeptides of thepresent invention comprise the amino acid sequenceMCVYIYVYTCMCVYIYVYICICVYIHVYTCICVYIHVYTCVCVYIYVYTCMCVYICIYVYIYICVCVSVYIYNRIIYILLALSL (SEQ ID NO: 440). Polynucleotidesencoding this polypeptide are also provided.

[0529] This gene is expressed primarily in the fetal liver/spleen, humanbrain, and retina.

[0530] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immune,neurologic, and visual diseases and/or disorders, particularlyretinoblastoma as well as other diseases or disorders involving theretina and/or brain. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theneurologic system and in eye development, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., immune, visual, retinal, neural,cancerous, and/or wounded tissues) or bodily fluids (e.g., serum,plasma, aqueous humor, vitreous humor, urine, amniotic fluid, synovialfluid and spinal fluid, vitreous and aqueous humors) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 198 as residues: Glu-48 toThr-54. Polynucleotides encoding said polypeptides are also provided.

[0531] The tissue distribution in fetal liver/spleen indicatespolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and diagnosis of hematopoietic related disorders suchas anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia sincestromal cells are important in the production of cells of hematopoieticlineages. Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Briefly, the uses include bone marrowcell ex-vivo culture, bone marrow transplantation, bone marrowreconstitution, radiotherapy or chemotherapy of neoplasia.

[0532] The gene product may also be involved in lymphopoiesis,therefore, it can be used in immune disorders such as infection,inflammation, allergy, immunodeficiency etc. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types.Alternatively, representative uses are described in the “Regeneration”and “Hyperproliferative Disorders” sections below, in Example 11, 15,and 18, and elsewhere herein. Briefly, the uses include, but are notlimited to the detection, treatment, and/or prevention of Alzheimer'sDisease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,panic disorder, learning disabilities, ALS, psychoses, autism, andaltered behaviors, including disorders in feeding, sleep patterns,balance, and perception. In addition, the gene or gene product may alsoplay a role in the treatment and/or detection of developmental disordersassociated with the developing embryo, sexually-linked disorders, ordisorders of the cardiovascular system.

[0533] Alternatively, expression of this gene with in the retina maysuggest gene is useful for the diagnosis, treatment, and/or preventionof a variety of eye disorders and/or conditions.

[0534] Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0535] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:77 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1578 of SEQID NO:77, b is an integer of 15 to 1592, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:77, and whereb is greater than or equal to a +14.

[0536] Features of Protein Encoded by Gene No: 68

[0537] The translation product of this gene shares sequence homologywith the glutamate-binding subunit of an N-methyl-D-asparate receptorcomplex. The amino acids L-glutamic and L-aspartic acids form the mostwidespread excitatory transmitter network in mammalian brain. Theexcitation produced by L-glutamic acid is important in the earlydevelopment of the nervous system, synaptic plasticity and memoryformation, seizures and neuronal degeneration. The receptors activatedby L-glutamic acid are a target for therapeutic intervention inneurodegenerative diseases, brain ischaemia and epilepsy. As such, theprotein product of this gene may also play a role in the regulation ofthe nitrous oxide synthase gene which is known to be a vital link invarious signal transduction pathways within the brain as well as othertissues (See Genbank No. bbs|61979 and Medline Article No.92049755).

[0538] Moreover, the translation product of this gene was also shown tohave homology to a neural membrane protein 35 (See Genbank Accession No.gb|AAC32463.11 (AF044201); all references available through thisaccession are hereby incorporated herein by reference; for example, Mol.Cell. Neurosci. 11 (5), 260-273 (1998)). The polypeptide of this genehas been determined to have two transmembrane domains at about aminoacid position 42-73, and 75-94 of the amino acid sequence referenced inTable 1 for this gene. Based upon these characteristics, it is believedthat the protein product of this gene shares structural features to IIIamembrane proteins.

[0539] When tested against U937 and Jurkat cell lines, supernatantsremoved from cells containing this gene activated the GAS (gammaactivating sequence) promoter element. Thus, it is likely that this geneactivates myeloid and T-cells, and to a lesser extent, other cells andtissue cell types, through the JAK-STAT signal transduction pathway. GASis a promoter element found upstream of many genes which are involved inthe Jak-STAT pathway. The Jak-STAT pathway is a large, signaltransduction pathway involved in the differentiation and proliferationof cells. Therefore, activation of the Jak-STAT pathway, reflected bythe binding of the GAS element, can be used to indicate proteinsinvolved in the proliferation and differentiation of cells.

[0540] Preferred polypeptides of the invention comprise the followingamino acid sequence: HASAWNLILLTVFTLS (SEQ ID NO: 441),VYAALGAGVFTLFLALDTQLLMGN (SEQ ID NO: 442), EEYIFGALNIYLDIIYIF (SEQ IDNO: 443), and/or WNLILLTVFTLSMAYLTGMLSSYYNT (SEQ ID NO: 444).Polynucleotides encoding these polypeptides are also provided.

[0541] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: MAYLTGMLSSYYNTTSVLLCLGITALVCLSVTVFSFQTKFDFTSCQGVLFVLLMTLFFSGLILAILLPFQYVPWLHAVYAALGAGVFTLFLALDTQLLMGNRRHSLSPEEYIFGALNIYLDIIYIFTFFLQLFGTNRE (SEQ ID NO: 445). Polynucleotidesencoding these polypeptides are also provided.

[0542] This gene is expressed primarily in the brain and to a lesserextent in dendritic cells and in the kidney cortex.

[0543] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,schizophrenia, epilepsy, brain ischaemia, and neurodegenerativediseases. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thenervous system expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,neural, cancerous and wounded tissues) or bodily fluids (e.g., serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO:199 as residues: Ala-12 toGlu-27, Pro-35 to Ser-43, Pro-70 to Gly-79, Ser-92 to Val-98, Pro-166 toLeu-175, Ser-234 to Thr-246. Polynucleotides encoding said polypeptidesare also provided.

[0544] The tissue distribution combined with the homology to a knownN-methyl-D-asparate receptor indicates polynucleotides and polypeptidescorresponding to this gene are useful for the detection, treatment,and/or prevention of neurodegenerative disease states, behavioraldisorders, or inflammatory conditions. Representative uses are describedin the “Regeneration” and “Hyperproliferative Disorders” sections below,in Example 11, 15, and 18, and elsewhere herein. Briefly, the usesinclude, but are not limited to the detection, treatment, and/orprevention of Alzheimer's Disease, Parkinson's Disease, Huntington'sDisease, Tourette Syndrome, meningitis, encephalitis, demyelinatingdiseases, peripheral neuropathies, neoplasia, trauma, congenitalmalformations, spinal cord injuries, ischemia and infarction, aneurysms,hemorrhages, schizophrenia, mania, dementia, paranoia, obsessivecompulsive disorder, depression, panic disorder, learning disabilities,ALS, psychoses, autism, and altered behaviors, including disorders infeeding, sleep patterns, balance, and perception. In addition, elevatedexpression of this gene product in regions of the brain indicates itplays a role in normal neural function. Potentially, this gene productis involved in synapse formation, neurotransmission, learning,cognition, homeostasis, or neuronal differentiation or survival. Thisprotein may play a role in the regulation of cellular division, and mayshow utility in the diagnosis, treatment, and/or prevention ofdevelopmental diseases and disorders. The protein can also be used togain new insight into the regulation of cellular growth andproliferation.

[0545] Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0546] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:78 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1565 of SEQID NO:78, b is an integer of 15 to 1579, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:78, and whereb is greater than or equal to a +14.

[0547] Features of Protein Encoded by Gene No: 69

[0548] The polypeptide of this gene has been determined to have atransmembrane domain at about amino acid position 37-62 of the aminoacid sequence referenced in Table 1 for this gene. Based upon thesecharacteristics, it is believed that the protein product of this geneshares structural features to Type Ia membrane proteins.

[0549] The translation product of this gene was also determined to havea conserved peroxidase-I domain (PROSITE entry PDOC00394; SwissInstitute of Bioinformatics) located at about amino acid position 15-25of the amino acid sequence referenced in Table 1 for this gene.

[0550] Preferred polypeptides of the invention comprise the followingamino acid sequence: TLSLLVSLHTV (SEQ ID NO: 446). Polynucleotidesencoding these polypeptides are also provided. Peroxidases areheme-binding enzymes that carry out a variety of biosynthetic anddegradative functions using hydrogen peroxide as the electron acceptor.Peroxidases are widely distributed throughout bacteria, fungi, plants,and vertebrates. In peroxidases the heme prosthetic group isprotoporphyrin 1× and the fifth ligand of the heme iron is a histidine(known as the proximal histidine). An other histidine residue (thedistal histidine) serves as an acid-base catalyst in the reactionbetween hydrogen peroxide and the enzyme. The regions around these twoactive site residues are more or less conserved in a majority ofperoxidases (see Dawson J. H., Science 240:433439(1988); Kimura S.,Ikeda-Saito M., Proteins 3:113-120(1988); Henrissat B., et al., Proteins8:251-257(1990); and Welinder K. G., Biochem. Biophys. Acta1080:215-220(1991). All references are hereby incorporated herein.)

[0551] This gene is expressed primarily in the brain.

[0552] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,neurological diseases and disorders, a non-limiting example of whichincludes, epilepsy. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thenervous system expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,neural, cancerous, and/or wounded tissues) or bodily fluids (e.g.,serum, plasma, urine, synovial fluid and spinal fluid) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

[0553] The tissue distribution in brain tissue indicates polynucleotidesand polypeptides corresponding to this gene are useful for thedetection, treatment, and/or prevention of neurodegenerative diseasestates, behavioral disorders, or inflammatory conditions. Representativeuses are described in the “Regeneration” and “HyperproliferativeDisorders” sections below, in Example 11, 15, and 18, and elsewhereherein. Briefly, the uses include, but are not limited to the detection,treatment, and/or prevention of Alzheimer's Disease, Parkinson'sDisease, Huntington's Disease, Tourette Syndrome, meningitis,encephalitis, demyelinating diseases, peripheral neuropathies,neoplasia, trauma, congenital malformations, spinal cord injuries,ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania,dementia, paranoia, obsessive compulsive disorder, depression, panicdisorder, learning disabilities, ALS, psychoses, autism, and alteredbehaviors, including disorders in feeding, sleep patterns, balance, andperception. In addition, elevated expression of this gene product inregions of the brain indicates it plays a role in normal neuralfunction. Potentially, this gene product is involved in synapseformation, neurotransmission, learning, cognition, homeostasis, orneuronal differentiation or survival. Furthermore, the protein may alsobe used to determine biological activity, to raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0554] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:79 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1382 of SEQID NO:79, b is an integer of 15 to 1396, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:79, and whereb is greater than or equal to a +14.

[0555] Features of Protein Encoded by Gene No: 70

[0556] When tested against Jurkat cell lines, supernatants removed fromcells containing this gene activated the GAS (gamma activating sequence)promoter element. Thus, it is likely that this gene activates T-cells,and to a lesser extent, other cells and tissue cell-types, through theJAK-STAT signal transduction pathway. GAS is a promoter element foundupstream of many genes which are involved in the Jak-STAT pathway. TheJak-STAT pathway is a large, signal transduction pathway involved in thedifferentiation and proliferation of cells. Therefore, activation of theJak-STAT pathway, reflected by the binding of the GAS element, can beused to indicate proteins involved in the proliferation anddifferentiation of cells. Additional embodiments of the inventioninclude polypeptides comprising the following amino acid sequences:MSSSGTSDASPSGSPVLASYKPAPPKDKLPETPRRRMKKSLSAPLHPEFEEVYRFGAESRKLLLREPVDAMPDPTPFLLARESAEVHLIKERPLVIPPIASDRSGEQHSPAREKPHKAHVGVAHRIHHATPPQPARGEDPGGRPGERRQGGEEALRDGQNCVKPAVPHPALSMHCEHHWEISATPFLFNPMHAKHFSHLPTHSPSASLALFFTPKYDRVPAAEYVFPNCCGQTPVCRIACF (SEQ ID NO: 447);MSSSGTSDASPSGSPVLASYKPAPPKDKLPETPRRRMKKSLSAPLHPEFEEVYRFGAESRKLLLREPVDAMPDPTPFLLARESAE (SEQ ID NO: 448);VHLIKERPLVIPPIASDRSGEQHSPAREKPHKAHVGVAHRIHHATPPQPARGED PGGRPGERR (SEQ IDNO: 449); QGGEEALRDGQNCVKPAVPHPALSMHCEHHWEISATPFLFNPMHAKHFSHLPTHSPSASLALFFTPKYDRVPAAEYVFPNCCGQTPVCRIACF (SEQ ID NO: 450);KRASQPPCTRNLKRSTDSGQRAGNSFCGNQWMLCPTPPHFCWLGSPPRSTSS KRGPSSS (SEQ ID NO:451); and PPSPPTEAASSTARPAKSRTRPTSGWHIGSTTPPRRSQPEVKTLAVDQVNGGKVVRKHSGTDRTV (SEQ ID NO: 452). Additional embodiments are directed topolynucleotides encoding these polypeptides.

[0557] The gene encoding the disclosed cDNA is believed to reside onchromosome 12. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 12.

[0558] This gene is expressed primarily in Endometrial Tumor, fetalliver, Hypothalamus, Larynx carcinoma III, Prostate Cancer.

[0559] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,endometrial tumor, larynx carcinoma III, prostate cancer, in addition toother proliferative diseases and/or disorders. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the reproductive, hepatic, and pulmonary systems,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types (e.g., hepatic,developmental, differentiating, proliferative, and cancerous, and/orother tissues) or bodily fluids (e.g., serum, plasma, urine, synovialfluid and spinal fluid, pulmonary surfactant) or another tissue or cellsample taken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.Preferred polypeptides of the present invention comprise immunogenicepitopes shown in SEQ ID NO: 201 as residues: Ala-62 to Tyr-71.Polynucleotides encoding said polypeptides are also provided.

[0560] The tissue distribution in tumors of endometrium, larynx, andprostate origins, combined with the detected GAS biological activity,indicates that polynucleotides and polypeptides corresponding to thisgene are useful for diagnosis and intervention of these tumors, inaddition to other tumors where expression has been indicated. Theexpression within cellular sources marked by proliferating cellsindicates this protein may play a role in the regulation of cellulardivision, and may show utility in the diagnosis, treatment, and/orprevention of developmental diseases and disorders, including cancer,and other proliferative conditions. Representative uses are described inthe “Hyperproliferative Disorders” and “Regeneration” sections below andelsewhere herein. Briefly, developmental tissues rely on decisionsinvolving cell differentiation and/or apoptosis in pattern formation.Alternatively, the tissue distribution within liver tissue indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for the detection and treatment of liver disorders and cancers(e.g. hepatoblastoma, jaundice, hepatitis, liver metabolic diseases andconditions that are attributable to the differentiation of hepatocyteprogenitor cells). In addition the expression in fetus would suggest auseful role for the protein product in developmental abnormalities,fetal deficiencies, pre-natal disorders and various would-healing modelsand/or tissue trauma. Furthermore, the protein may also be used todetermine biological activity, to raise antibodies, as tissue markers,to isolate cognate ligands or receptors, to identify agents thatmodulate their interactions, in addition to its use as a nutritionalsupplement. Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tissues.

[0561] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:80 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1216 of SEQID NO:80, b is an integer of 15 to 1230, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:80, and whereb is greater than or equal to a +14.

[0562] Features of Protein Encoded by Gene No: 71

[0563] In another embodiment, polypeptides of the invention comprise thefollowing amino acid sequence:MWNPNAGQPGPNPYPPNIGCPGGSNPAHPPPINPPFPPGPCPPPPGAPHGNPAFPPGGPPHPVPQPGYPGCQPLGPYPPPYPPPAPGIPPVNPLAPGMVGPAVIVDKKMQKKMKKAHKKMHKHQKHHKYHKHGKHSSSSSSSSSSDSD (SEQ ID NO: 453);RVGPDAWADAWEQAQAAVERLEDTPKHVESQCRAARAKSISPQYWVPWRF QSCPPTTY (SEQ ID NO:454); STLSPRPLSSSPRSSPWQSSFPPRWAPSSCATARVSRMPTVGSLPSSIPTACPWNPSCESLGSWHGWTSSDSRQEDAEENEESS (SEQ ID NO: 455);MPGSQGQIHIPPILGALEVPILPTHELLIHPFPQAPVLLPQELPMAIQLSPQVGPLILCHSQGIQDANRWVPTLLHTHRLPLESLL (SEQ ID NO: 456); and/orMASIPPLPPPLPAVILTEYRPWTLPSSLTSSALPSSFRCHVVLGECSPCAPHPLP XPEPHPAVEP (SEQID NO: 457). Polynucleotides encoding these polypeptides are alsoprovided.

[0564] This gene is expressed primarily in bone marrow and primarydendritic cells, in addition to macrophages.

[0565] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis of immuneand haematopoeitic diseases and/or disorders. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune, expression of this gene at significantlyhigher or lower levels may be routinely detected in certain tissues orcell types (e.g., haematopoeitic, immune, and cancerous, and/or othertissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluidand spinal fluid, and/or lymph) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0566] The tissue distribution in bone marrow indicates polynucleotidesand polypeptides corresponding to this gene are useful for the treatmentand diagnosis of hematopoietic related disorders such as anemia,pancytopenia, leukopenia, thrombocytopenia or leukemia since stromalcells are important in the production of cells of hematopoieticlineages. Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Briefly, the uses include bone marrowcell ex-vivo culture, bone marrow transplantation, bone marrowreconstitution, radiotherapy or chemotherapy of neoplasia.

[0567] The gene product may also be involved in lymphopoiesis,therefore, it can be used in immune disorders such as infection,inflammation, allergy, immunodeficiency etc. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Furthermore,the protein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0568] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:81 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1125 of SEQID NO:81, b is an integer of 15 to 1139, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:81, and whereb is greater than or equal to a +14.

[0569] Features of Protein Encoded by Gene No: 72

[0570] The translated product of this gene is likely a Type Ia membraneprotein containing a transmembrane domain at about amino acid residues125 through about 141.

[0571] In another embodiment, polypeptides of the invention comprise thefollowing amino acid sequence:PRHTYWGIWLVPAAMASPHSHPAQGVLQPPGPQPRWEDRVALGTRGRSPGAYLTESAPQQASTTPGPPTCHGKVGSEWAWLGAAPGPLPTHPSHYAIRVPSNICSCPGASSAPALRGVVRQPPGPQNPRQGGRRGTRASPVGSLFCV (SEQ ID NO: 458);MFAVLPAVEGRATPHQDRTCYPSRSRPWPSQPSPRGSMPVPRPGAARGQLDGHVQGQGWALQWGGPPAPAVYRRMALPPRAAGSYLDRKCPHPLPGARLCPG LPL (SEQ ID NO: 459);VFGAVFLTTPSHDLATPTGASGWCLLPWPAPTLTLHRGSCSPQAHSLVGRTGWPWGQEGGAQGLTSLRVLPSRHPLPQGPPHVMARLVVNGPGWEQPLAHCPPTHLTMQFEFQATFAPALGPALPQP (SEQ ID NO: 460);HEEPPAGFGLRSLWRRSPPHEVGARLPNGAFGFSVRCLLCFPPWRAEPPHIRIGRATPPGPGPGPASPALEARCLCQGQGQPEGSWMATCRVKAGPCSGAGRQPQQFTDAWLFLPEQPAATWTGNVLIPSLGPGSALAFLCEPLLSLCCLGTPDRGVRVCPSVTFYSPRVEERKRGKSKGVQTPPQ (SEQ ID NO: 461);MATCRVKAGPCSGAGRQPQQFTDAWLFLPEQPAATWTGNVLIPSLGPGSALAFLCEPLLSLCCLGTPDRGVRVCPSVTFYSPRVEERKRGKSKGVQTPPQ (SEQ ID NO: 462);MKWFSTQPLWLNTKQRSHRRGPGPPPAPLSGVLGSRGLPHHPSQGWGRAGPRAGANVAWNSNCIVRWVGGQWARGCSQPGPFTTNLAMTCGGPWGSGCLLGSTLSEVSPWAPPSCPQGHPVLPTRLWAWGLQDPLCRVRVGAGHGSRHQPDAPVGVARSWDGVVRNTAPKTQNKNTTNGRRSPPPMEVGFEPLLIFPVSFLQPLVSRKSQTGTHAHHGQESRDSTKKGGVHRGRPGQSLAPGRG (SEQ ID NO: 463);KVTDGHTRTPRSGVPRQHKERRGSQRKARAEPGPREGMRTFPVQVAAGCSGRKSHASVNCWGWRPAPLQGPALTLHVAIQLPSGCPWPWHRHRASRAGLAGPGPGPGGVARPILMWGGSALHGGKHSKHRTLKPKAPLGSLAPTSWGGDRRHR DLSPKPAGGSSC (SEQ IDNO: 464); and/or MRTFPVQVAAGCSGRKSHASVNCWGWRPAPLQGPALTLHVAIQLPSGCPWPWHRHRASRAGLAGPGPGPGGVARPILMWGGSALHGGKHSKHRTLKPKAPLGSLAPTSWGGDRRHRDLSPKPAGGSSC (SEQ ID NO: 465). Polynucleotides encodingthese polypeptides are also provided.

[0572] The gene encoding the disclosed cDNA is believed to reside onchromosome 7. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 7.

[0573] This gene is expressed primarily in healing wound tissues,macrophage-oxLDL, hemangiopericytoma, and CD34+ cells.

[0574] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, healingwound, and proliferative diseases and/or disorders, particularly softtissue cancers, such as hemangiopericytoma. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of healing wounds, expression of this gene at significantlyhigher or lower levels may be routinely detected in certain tissues orcell types (e.g., lymph, cancerous, and/or wounded tissues) or bodilyfluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid,and/or lymph) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder. Preferred polypeptides of thepresent invention comprise immunogenic epitopes shown in SEQ ID NO: 203as residues: Met-1 to Gly-6, Arg-23 to Gly-33, Arg-60 to Ala-66, Thr-90to Gly-103, Glu-105 to Trp-112. Polynucleotides encoding saidpolypeptides are also provided.

[0575] The tissue distribution within healing wounds indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of cancer and other proliferativedisorders. Representative uses are described elsewhere herein.Expression within cellular sources marked by proliferating cellsindicates that this protein may play a role in the regulation ofcellular division. Additionally, the expression in hematopoietic cellsand tissues indicates that this protein may play a role in theproliferation, differentiation, and/or survival of hematopoietic celllineages. In such an event, this gene may be useful in the treatment oflymphoproliferative disorders, and in the maintenance anddifferentiation of various hematopoietic lineages from earlyhematopoietic stem and committed progenitor cells. Similarly, embryonicdevelopment also involves decisions involving cell differentiationand/or apoptosis in pattern formation. Thus this protein may also beinvolved in apoptosis or tissue differentiation and could again beuseful in cancer therapy. Furthermore, the protein may also be used todetermine biological activity, to raise antibodies, as tissue markers,to isolate cognate ligands or receptors, to identify agents thatmodulate their interactions, in addition to its use as a nutritionalsupplement. Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tissues.

[0576] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:82 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1395 of SEQID NO:82, b is an integer of 15 to 1409, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:82, and whereb is greater than or equal to a +14.

[0577] Features of Protein Encoded by Gene No: 73

[0578] The translation product of this gene has homology to thePro-Pol-dUTPase polyprotein of a newly discovered retrovirus. Since thisprotein also shares homology to the human HERV-L element, andconsidering that most retroviruses integrate their proviral form intoeukaryotic genomes through a homologous recombination mechanism, thisgene is useful in providing protection against retroviral infections orcould be used in the development of gene therapy applications (SeeGenbank Accession No.2065210; all references available through thisaccession are hereby incorporated by reference herein).

[0579] Preferred polypeptides of the invention comprise the followingamino acid sequence: GLMECLIHRHGSH (SEQ ID NO: 466), and/orSTKGMQFILTGITLSGY (SEQ ID NO: 467). Polynucleotides encoding thesepolypeptides are also provided.

[0580] This gene is expressed primarily in CD34 positive cells.

[0581] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immunediseases and/or disorders, particularly viral infections. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., immune, and cancerous, wounded,and/or other tissues) or bodily fluids (e.g., serum, plasma, urine,synovial fluid and spinal fluid, and/or lymph) or another tissue or cellsample taken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.Preferred polypeptides of the present invention comprise immunogenicepitopes shown in SEQ ID NO: 204 as residues: Arg-39 to Thr-49, Leu-52to Gly-60, Ser-67 to Arg-76, Gln-130 to Phe-137, Ser-139 to His-148.Polynucleotides encoding said polypeptides are also provided.

[0582] The tissue distribution in CD34+ immune cells combined with thehomology to a retroviral protein indicates that polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosis andtreatment of a variety of immune system disorders. Expression of thisgene product in immune indicates a role in the regulation of theproliferation; survival; differentiation; and/or activation ofpotentially all hematopoietic cell lineages, including blood stem cells.This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer e.g. by boosting immuneresponses. Expression in cells of lymphoid origin, the natural geneproduct may be involved in immune functions. Therefore it may be alsoused as an agent for immunological disorders including arthritis,asthma, immune deficiency diseases such as AIDS, and leukemia. Inaddition, this gene product may have commercial utility in the expansionof stem cells and committed progenitors of various blood lineages, andin the differentiation and/or proliferation of various cell types.Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0583] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:83 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 700 of SEQID NO:83, b is an integer of 15 to 714, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:83, and where bis greater than or equal to a +14.

[0584] Features of Protein Encoded by Gene No: 74

[0585] The translation product of this gene shares sequence homologywith mouse, bovine, and human butyrophilins, which are thought to beimportant in lactation especially during the latter part of pregnancy.Butyrophilin is a glycoprotein of the immunoglobulin superfamily that issecreted in association with the milk-fat-globule membrane from mammaryepithelial cells (See Genbank Accession No.gb|AAB51034.1, and GeneseqAccession No. W97814; all references available through these accessionsare hereby incorporated herein by reference; for example, Mamm. Genome 7(12), 900-905 (1996)). Based on the sequence similarity, the translationproduct of this gene is expected to share at least some biologicalactivities with glycoproteins. Such activities are known in the art,some of which are described elsewhere herein.

[0586] In another embodiment, polypeptides of the invention comprise thefollowing amino acid sequence:PRVRALLFARSLRLCRWGAKRLGVASTEAQRGVSFKLEEKTAHSSLALFRDDTGVKYGLVGLEPTKVALNVERFREWAVVLADTAVTSGRHYWEVTVKRSQQFRIGVADVDMSRDSCIGVDDRSWVFTMPSASGTPCWPTRKPQLRVLGSQEVGLLLEYEAQKLSLVDVSQVSVVHTLQTDFRGPVVPAFALWDGELLTHSGLEVP EGL (SEQ ID NO:468), and/or MSRDSCIGVDDRSWVFTMPSASGTPCWPTRKPQLRVLGSQEVGLLLEYEAQKLSLVDVSQVSVVHTLQTDFRGPVVPAFALWDGELLTHSGLEVPEGL (SEQ ID NO: 469).Polynucleotides encoding these polypeptides are also provided.

[0587] This gene is expressed primarily in adult heart, LNCAP cell line,OB cell line (HOS fraction), and epididymis, and to a lesser extent in avariety of other cells and tissues.

[0588] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, coronarydisease and heart tumors and reproductive disorders, particularly thoseof the male reproductive system. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularlythose of the heart and reproductive system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., cardiovascular, cardiac,reproductive, and cancerous and wounded tissues) or bodily fluids (e.g.,serum, plasma, urine, seminal fluid, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder. Preferred polypeptides of the present inventioncomprise immunogenic epitopes shown in SEQ ID NO: 205 as residues:Gly-30 to Ser-36. Polynucleotides encoding said polypeptides are alsoprovided.

[0589] The tissue distribution and homology to butyrophilin indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for determining the mechanisms underlying mammary-specific geneexpression, lactation, and potentially for the production of copiousamounts of butyrophilin or heterologous proteins in the milk oftransgenic animals. The secreted protein can also be used to determinebiological activity, to raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, and as nutritional supplements. It may also have a verywide range of biological activities. Representative uses are describedin the “Chemotaxis” and “Binding Activity” sections below, in Examples11, 12, 13, 14, 15, 16, 18, 19, and 20, and elsewhere herein. Briefly,the protein may possess the following activities: cytokine, cellproliferation/differentiation modulating activity or induction of othercytokines; immunostimulating/immunosuppressant activities (e.g. fortreating human immunodeficiency virus infection, cancer, autoimmunediseases and allergy); regulation of hematopoiesis (e.g. for treatinganemia or as adjunct to chemotherapy); stimulation or growth of bone,cartilage, tendons, ligaments and/or nerves (e.g. for treating wounds,stimulation of follicle stimulating hormone (for control of fertility);chemotactic and chemokinetic activities (e.g. for treating infections,tumors); hemostatic or thrombolytic activity (e.g. for treatinghemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g.for treating septic shock, Crohn's disease); as antimicrobials; fortreating psoriasis or other hyperproliferative diseases; for regulationof metabolism, and behavior. Also contemplated is the use of thecorresponding nucleic acid in gene therapy procedures. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0590] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:84 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1083 of SEQID NO:84, b is an integer of 15 to 1097, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:84, and whereb is greater than or equal to a +14.

[0591] Features of Protein Encoded by Gene No: 75

[0592] The translation product of this gene shares sequence homologywith angiopoietin-2 which is thought to be important in regulation ofangiogenesis through the Tie2, or other receptor tyrosine kinase (SeeGenbank Accession Nos. gb|AAC97965.11 (AF110520), and gb|AAB63189.11(AF004326); in addition to Geneseq Accession No. R94603; all referencesavailable through these accessions are hereby incorporated herein byreference; for example, Science 277 (5322), 55-60 (1997)). Based on thesequence similarity, the translation product of this gene is expected toshare at least some biological activities with angiogenic and kinaseproteins. Such activities are known in the art, some of which aredescribed elsewhere herein.

[0593] In another embodiment, polynucleotides of the invention comprisethe following nucleic acid sequence:GCACGAGCGGCACGAGCGGATCCTCACACGACTGTGATCCGATTCTTTCCAGCGGCTTCTGCAACCAAGCGGGTCTTACCCCCGGTCCTCCGCGTCTCCAGTCCTCGCACCTGGAACCCCAACGTCCCCGAGAGTCCCCGAATCCCCGCTCCCAGGCTACCTAAGAGGATGAGCGGTGCTCCGACGGCCGGGGCAGCCCTGATGCTCTGCGCCGCCACCGCCGTGCTACTGAGCGCTCAGGGCGGACCCGTGCAGTCCAAGTCGCCGCGCTTTGCGTCCTGGGACGAGATGAATGTCCTGGCGCACGGACTCCTGCAGCTCGGCCAGGGGCTGCGCGAACACGCGGAGCGCACCCGCAGTCAGCTGAGCGCGCTGGAGCGGCGCCTGAGCGCGTGCGGGTCCGCCTGTCAGGGAACCGAGGGGTCCACCGACCTCCCGTTAGCCCCTGAGAGCCGGGTGGACCCTGAGGTCCTTCACAGCCTGCAGACACAACTCAAGGCTCAGAACAGCAGGATCCAGCAACTCTTCCACAAGGTGGCCCAGCAGCAGCGGCACCTGGAGAAGCAGCACCTGCGAATTCAGCATCTGCAAAGCCAGTTTGGCCTCCTGGACCACAAGCACCTAGACCATGAGGTGGCCAAGCCTGCCCGAAGAAAGAGGCTGCCCGAGATGGCCCAGCCAGTTGACCCGGCTCACAATGTCAGCCGCCTGCACCGGCTGCCCAGGGATTGCCAGGAGCTGTTCCAGGTTGGGGAGAGGCAGAGTGGACTATTTGAAATCCAGCCTCAGGGGTCTCCGCCATTTTTGGTGAACTGCAAGATGACCTCAGATGGAGGCTGGACAGTAATTCAGAGGCGCCACGATGGCTCAGTGGACTTCAACCGGCCCTGGGAAGCCTACAAGGCGGGGTTTGGGGATCCCCACGGCGAGTTCTGGCTGGGTCTGGAGAAGGTGCATAGCATCACGGGGGACCGCAACAGCCGCCTGGCCGTGCAGCTGCGGGACTGGGATGGCAACGCCGAGTTGCTGCAGTTCTCCGTGCACCTGGGTGGCGAGGACACGGCCTATAGCCTGCAGCTCACTGCACCCGTGGCCGGCCAGCTGGGCGCCACCACCGTCCCACCCAGCGGCCTCTCCGTACCCTTCTCCACTTGGGACCAGGATCACGACCTCCGCAGGGACAAGAACTGCGCCAAGAGCCTCTCTGGAGGCTGGTGGTTTGGCACCTGCAGCCATTCCAACCTCAACGGCCAGTACTTCCGCTCCATCCCACAGCAGCGGCAGAAGCTTAAGAAGGGAATCTTCTGGAAGACCTGGCGGGGCCGCTACTACCCGCTGCAGGCCACCACCATGTTGATCCAGCCCATGGCAGCAGAGGCAGCCTCCTAGCGTCCTGGCTGGGCCTGGTCCCAGGCCCACGAAAGACGGTGACTCTTGGCTCTGCCCGAGGATGTGGCCGTTCCCTGCCTGGGCAGGGGCTCCAAGGAGGGGCCATCTGGAAACTTGTGGACAGAGAAGAAGACCACGACTGGAGAAGCCCCCTTTCTGAGTGCAGGGGGGCTGCATGCGTTGCCTCCTGAGATCGAGGCTGCAGGATATGCTCAGACTCTAGAGGCGTGGACCAAGGGGCATGGAGCTTCACTCCTTGCTGGCCAGGGAGTTGGGGACTCAGAGGGACCACTTGGGGCCAGCCAGACTGGCCTCAATGGCGGACTCAGTCACATTGACTGACGGGGACCAGGGCTTGTGTGGGTCGAGAGCGCCCTCATGGTGCTGGTGCTGTTGTGTGTAGGTCCCCTGGGGACACAAGCAGGCGCCAATGGTATCTGGGCGGAGCTCACAGAGTTCTTGGAATAAAAGCAACCTCAGAACAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAA (SEQ IDNO:470), and/or ATGAGCGGTGCTCCGACGGCCGGGGCAGCCCTGATGCTCTGCGCCGCCACCGCCGTGCTACTGAGCGCTCAGGGCGGACCCGTGCAGTCCAAGTCGCCGCGCTITGCGTCCTGGGACGAGATGAATGTCCTGGCGCACGGACTCCTGCAGCTCGGCCAGGGGCTGCGCGAACACGCGGAGCGCACCCGCAGTCAGCTGAGCGCGCTGGAGCGGCGCCTGAGCGCGTGCGGGTCCGCCTGTCAGGGAACC GAGGGGTCCACCGACCTCCCGTTAGCCCCTGAGAGCCGGGTGGACCCTGAGGTCCTTCACAGCCTGCAGACACAACTCAAGGCTCAGAACAGCAGGATCCAGCAACTCTTCCACAAGGTGGCCCAGCAGCAGCGGCACCTGGAGAAGCAGCACCTGCGAATTCAGCATCTGCAAAGCCAGTTTGGCCTCCTGGACCACAAGCACCTAGACCATGAGGTGGCCAAGCCTGCCCGAAGAAAGAGGCTGCCCGAGATGGCCCAGCCAGTTGACCCGGCTCACAATGTCAGCCGCCTGCACCGGCTGCCCAGGGATTGCCAGGAGCTGTTCCAGGTTGGGGAGAGGCAGAGTGGACTATTTGAAATCCAGCCTCAGGGGTCTCCGCCATTTTTGGTGAACTGCAAGATGACCTCAGATGGAGGCTGGACAGTAATTCAGAGGCGCCACGATGGCTCAGTGGACTTCAACCGGCCCTGGGAAGCCTACAAGGCGGGGTTTGGGGATCCCCACGGCGAGTTCTGGCTGGGTCTGGAGAAGGTGCATAGCATCACGGGGGACCGCAACAGCCGCCTGGCCGTGCAGCTGCGGGACTGGGATGGCAACGCCGAGTTGCTGCAGTTCTCCGTGCACCTGGGTGGCGAGGACACGGCCTATAGCCTGCAGCTCACTGCACCCGTGGCCGGCCAGCTGGGCGCCACCACCGTCCCACCCAGCGGCCTCTCCGTACCCTTCTCCACTTGGGACCAGGATCACGACCTCCGCAGGGACAAGAACTGCGCCAAGAGCCTCTCTGGAGGCTGGTGGTTTGGCACCTGCAGCCATTCCAACCTCAACGGCCAGTACTTCCGCTCCATCCCACAGCAGCGGCAGAAGCTTAAGAAGGGAATCTTCTGGAAGACCTGGCGGGGCCGCTACTACCCGCTGCAGGCCACCACCATGTTGATCCAGCCCATGGCAGCAGAGGCAGCCTCCTAG (SEQ ID NO:471). A preferred polypeptidefragment of the invention comprises the following amino acid sequence:MAQWTSTGPGKPTRRGLGIPTASSGWVWRRC (SEQ ID NO: 472)IASWGTATAAWPCSCGTGMATPSCCSSPCTWVARTRPIACSSLHPWPASWAPPPSHPAASPYPSPLGTRITTSAGTRTAPRASLEAGGLAPAAIPTFNGPVLPAPSHSSGRSLRRESSGRPAGRYYPLQATTMLIQPMAA EAAS.

[0594] Polynucleotides encoding these polypeptides are also provided.The translated product of this gene contains a Fibrinogen beta and gammachains C-terminal domain signature (consensus pattern WW[LMFYW]. {2}C.{2} [GSA]. {2}NG; PROSITE entry PDOC00445, Swiss Institute ofBioinformatics). Fibrinogen, the principal protein of vertebrate bloodclotting is an hexamer containing two sets of three different chains(alpha, beta, and gamma), linked to each other by disulfide bonds. TheN-terminal sections of these three chains are evolutionary related andcontain the cysteines that participate in the cross-linking of thechains. However, there is no similarity between the C-terminal part ofthe alpha chain and that of the beta and gamma chains. The C-terminalpart of the beta and gamma chains forms a domain of about 270 amino-acidresidues. Fibrinogen beta and gamma chains C-terminal domains may beimportant for regulating protein-protein interactions. For references onthese domains see Doolittle R. F., Annu. Rev. Biochem. 53:195-229(1984)and Xu X., and Doolittle R. F., Proc. Natl. Acad. Sci. U.S.A.87:2097-2101(1990); all references are hereby incorporated herein byreference.

[0595] Preferred polypeptides of the invention comprise the amino acidsequences: WWFGTCSHSNLNG (SEQ ID NO: 473) and SGGWWFGTCSHSNLNGQYF (SEQID NO: 474). Also preferred are the polynucleotides encoding thesepolypeptides.

[0596] The gene encoding the disclosed cDNA is believed to reside onchromosome 19. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 19.

[0597] This gene is expressed primarily in oseteoarthritic tissues,kidney cortex, bone marrow, larynx carcinoma, and pineal gland, and to alesser extent in placenta, stromal cells, epithelioid sarcoma, and avariety of other cells and tissues.

[0598] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,arthritis, kidney and urinary tract disorders, immune cell and systemdysfunctions, disorders of the pineal gland and brain, and carcinomas,particularly of the larnyx. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularlythose of the immune, connective, endocrine, and urinary systems,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types (e.g., cancerous andwounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO: 206 as residues: Pro-27 to Arg-34, Glu-60 to Gln-65, Cys-80 toThr-87, Leu-109 to Ile-116, Ala-124 to Gln-133, Lys-158 to Leu-165,Arg-229 to Ser-234, Asp-236 to Trp-241, Thr-266 to Ser-271, Thr-328 toLys-343, Ser-355 to Tyr-363, Ile-367 to Lys-376, Thr-382 to Tyr-387.Polynucleotides encoding said polypeptides are also provided.

[0599] The tissue distribution and homology to angiopoietin-2 indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for the regulation of angiogenesis, particularly sinceangiogenesis is thought to depend on a precise balance of positive andnegative regulation. Angiopoietin-1 (Angl) is an angiogenic factor thatsignals through the endothelial cell-specific Tie2 receptor tyrosinekinase and, like vascular endothelial growth factor, is essential fornormal vascular development in the mouse. Angiopoietin-2 is a naturallyoccurring antagonist for Angiopoietin-1 and Tie2. Transgenicoverexpression of Angiopoietin-2 disrupts blood vessel formation in themouse embryo. In adult mice and humans, Angiopoietin-2 is expressed onlyat sites of vascular remodeling. As such, this gene, or antagoniststhereof, are useful in the diagnosis and treatment of arthritis, bonegrowth and remodeling, cancers (particularly those of bone, connective,lymphatic, and vascular tissues), ischaemia, lymphangiogenesis,lymphadnitis, lymphadenoma, lymphadenosis, Iymphangitis,lymphangioendothelioma, lymphangioma, lymphangiophlebitis,lymphangiosarcom, Iymphatitis, lymphedema, lymphenteritis, angioma,angiomegaly, amgiomyosarcoma, amgiomyoma, angiomyolipoma,angiomyoneuroma, angioneuromyoma, angiosarcoma, angiostenosis,angiotelectasis, and as a lymphagogue. Furthermore, the protein may alsobe used to determine biological activity, to raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0600] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:85 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1917 of SEQID NO:85, b is an integer of 15 to 1931, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:85, and whereb is greater than or equal to a +14.

[0601] Features of Protein Encoded by Gene No: 76

[0602] The translation product of this gene was shown to have homologyto the DPM2 mannosyl transferase gene, which is known to be important inO-linked oligosaccaride glycosylation of proteins. Mutations within thisgene have been shown to result in reduced levels of O-linkedglycosylation. Since defects in proper protein glycosylation can resultin the development of antigen-specific antibodies to such protein oraltered pharmacokinetics (i.e., plasma half-life, in vivo clearancerate, etc.), the protein product of this gene may show utility in thetreatment, diagnosis, and/or prevention of various abnormalitiesinvolving oligosaccaride metabolism, specifically those associated withO-glycosylation (See Genbank Accession No.R47201).

[0603] Preferred polypeptides of the invention comprise the followingamino acid sequence: GHDLPQDAWLRWVLAGALCAGGWAVNYLPFFL (SEQ ID NO: 475),and/or FLYHYLPALTFQILLLPV (SEQ ID NO: 476). Polynucleotides encodingthese polypeptides are also provided.

[0604] The gene encoding the disclosed cDNA is believed to reside onchromosome 9. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 9.

[0605] This gene is expressed primarily in brain and melanocytes and toa lesser extent in breast, testis, and colon.

[0606] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, cancers,particularly of the brain and melanocyte, in addition to neurologicaldisorders. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thebrain, central nervous system, PNS, epithelial tissues including otherparts of the integumentary system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., neural, cancerous and woundedtissues) or bodily fluids (e.g. lymph, serum, plasma, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO: 207 as residues: His-31 to Gln-38, Tyr-65 to Ser-71. Polynucleotidesencoding said polypeptides are also provided.

[0607] The tissue distribution in brain tissue, combined with thehomology to a known enzyme involved in oligosaccaride metabolism,indicates polynucleotides and polypeptides corresponding to this geneare useful for the detection, treatment, and/or prevention ofneurodegenerative disease states, behavioral disorders, or inflammatoryconditions. Representative uses are described in the “Regeneration” and“Hyperproliferative Disorders” sections below, in Example 11, 15, and18, and elsewhere herein. Briefly, the uses include, but are not limitedto the detection, treatment, and/or prevention of Alzheimer's Disease,Parkinson's Disease, Huntington's Disease, Tourette Syndrome,meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0608] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:86 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1078 of SEQID NO:86, b is an integer of 15 to 1092, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:86, and whereb is greater than or equal to a +14.

[0609] Features of Protein Encoded by Gene No: 77

[0610] Preferred polypeptides of the invention comprise the followingamino acid sequence: DICRLERAVCRDEPSALARALTWRQARAQAGA (SEQ ID NO: 478),XAPATXAWDTVVPPLPRKCQCSGSARSHGAGRSALHSPLEGSRPKVPAGAVGKSLPGQSRPQHCLPPKQPKQCRPGLELKEGPLLTPTRASVQLSHPACLYWAPL LWIRDPASV (SEQ IDNO: 479), XAPATXAWDTVVPPLPRKCQCSGSARSHGAGRSALHSPLEGSRPKVPAGAVG KSL (SEQID NO: 480), PGQSRPQHCLPPKQPKQCRPGLELKEGPLLTPTRASVQLSHPACLYWAPLLWIRDPASV (SEQ ID NO: 481), and/orMSPLPWPGPLPGGRQGHRLEPCCSSGCAGGPTWPHCSSQSWPMXSARHXGL GHCCPSSP (SEQ ID NO:477). Polynucleotides encoding these polypeptides are also provided.

[0611] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: DICRLERAVCRDEPSALARALTWRQARAQAGAMLLFGLCWGPYVATLLLSVLAYXQRPPLXPGTLLSLLS LGSASAAAVPVAMGLGDQRYTAPWRAAAQRCLQGLWGRASRDSPGPSIAYHPSSQSSVDLDLN (SEQ ID NO: 482). Polynucleotides encoding thesepolypeptides are also provided.

[0612] This gene is expressed primarily in cells of the immune system,including dendritic cells and T cells.

[0613] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, diseasesand/or disorders affecting the immune system, particularlyimmunodeficiencies such as AIDS. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe immune system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., immune, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder.

[0614] The tissue distribution in dendritic and T cells indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis, treatment and/or prevention of a variety of immunesystem disorders. Representative uses are described in the “ImmuneActivity” and “Infectious Disease” sections below, in Example 11, 13,14, 16, 18, 19, 20, and 27, and elsewhere herein. Expression of thisgene product in tonsils indicates a role in the regulation of theproliferation; survival; differentiation; and/or activation ofpotentially all hematopoietic cell lineages, including blood stem cells.This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer e.g., by boosting immuneresponses. Expression in cells of lymphoid origin, the natural geneproduct may be involved in immune functions. Therefore it may be alsoused as an agent for immunological disorders including arthritis,asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoidarthritis, granulomatous disease, inflammatory bowel disease, sepsis,acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such asT-cell mediated cytotoxicity; immune reactions to transplanted organsand tissues, such as host-versus-graft and graft-versus-host diseases,or autoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, scierodermaand tissues.

[0615] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Furthermore,the protein may also be used to determine biological activity, raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0616] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:87 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 564 of SEQID NO:87, b is an integer of 15 to 578, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:87, and where bis greater than or equal to a +14.

[0617] Features of Protein Encoded by Gene No: 78

[0618] Preferred polypeptides of the invention comprise the followingamino acid sequence: MERVGMESGEMVCGLGSACNNPSDLGQVPVPLWXSVSPPVFGXGWNGH,(SEQ ID NO: 483) MRSFQDVSALEEWRGGKDLEPTHSLLLLLPLRDLLVVLGEIRKRQMEGCVW(SEQ ID NO: 484) KGWGWNPEKWFAVLALPVTTRVTLGKSLSLSGXQFLHLYLERVGMGTEVLSSSDLL, MHPAGPTFMGSKPIREQQFGPDACLLLLCVAMAGTEASRAAQQCTSQKVRA (SEQ ID NO:485) GQDFSAHSNPXQIQVEKLXPREGQGLAQGHSGCYRQSQDRKPFLRIPSPPFPYTTLHLPFPDFAKNH, MHPAGPTFMGSKPIREQQFGPDACLLLLCVAMAGTEASRAAQQCTSQKVRA (SEQID NO: 486) GQDFSAHSNP,PREGQGLAQGHSGCYRQSQDRKPFLRIPSPPFPYTTLHLPFPDFAKNH, (SEQ ID NO: 487)DPRVRKPPTATLTTARTRPTTTD, and/or (SEQ ID NO: 488)AALEASVPAIATQRSSRQASGPNCCSLMGLDPMKVGPAGCISWDSVEADQVA (SEQ ID NO: 489)GASGGRIEVKGCGMENLXRLHLGSGKGQXX.

[0619] Polynucleotides encoding these polypeptides are also provided.

[0620] This gene is expressed primarily in prostate and gall bladder.

[0621] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, disordersaffecting the reproductive and gastrointestinal systems, includingcancer. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thereproductive and urogenital systems, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., reproductive, cancerous and woundedtissues) or bodily fluids (e.g., lymph, bile, seminal fluid, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 209 as residues: Arg-21 toGlu-30. Polynucleotides encoding said polypeptides are also provided.

[0622] The tissue distribution in gall bladder indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis, prevention, and/or treatment of various metabolicdisorders such as Tay-Sachs disease, phenylkenonuria, galactosemia,porphyrias, and Hurler's syndrome. In addition, expression of this geneproduct in the prostate—while likely to be reflective of non-specificexpression of a variety of genes in the testes—may nevertheless beindicative of a role for this gene product in normal prostate function,and may implicate this gene product in male fertility, and could evensuggest its use as a male contraceptive. Furthermore, the protein mayalso be used to determine biological activity, raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0623] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:88 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 685 of SEQID NO:88, b is an integer of 15 to 699, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:88, and where bis greater than or equal to a +14.

[0624] Features of Protein Encoded by Gene No: 79

[0625] Preferred polypeptides of the invention comprise the followingamino acid sequence:GXANPEDSVCILEGFSVTALSILQHLVCHSGAVRLPITVRSGGRFCCWGRKQE PGSQXSDGD (SEQ IDNO: 491), AVQQQHRVPQTAHCPPLLVGPWGSPCPPHCQPLSVQHHRERSDHLHITLAVGASDWGQGALAHQA (SEQ ID NO: 492),PKTLPVISCPGSSVCSKCCQSASAQRHPCLACCWLLSSSPCWRTTTSWHLSSVPTQKAASCCCCTCTSHHGLTEWPWRHNGSSWNKRWCGSWLSLVCKSPLPPVTGSNCQCNVEVVRALTVMLHRQWLTVRRAGGPPRTDQQRRTVRCLRDTVLLLHGLSQKDKLFMMHCVEVLHQFDQVMPGVSMLIRGLPDVTDCEEAALDDLC AAETDVEDPEVECG (SEQID NO: 493), QSPLPPVTGSNCQCNVEVVRALTVMLHRQWLTVRRAGGPPRTDQQRRTVRCLRDTVLLLHGLSQKDKLFMMHCVEVLHQFDQVMPGVSMLIRGLPDVTDCEEAALDDLCAAETDVEDPEVECG (SEQ ID NO: 495),QSPLPPVTGSNCQCNVEVVRALTVMLHRQWLTVRRAGGPPRTDQQRRTVRC LRDTVLLLHGLS (SEQ IDNO: 496), QKDKLFMMHCVEVLHQFDQVMPGVSMLIRGLPDVTDCEEAALDDLCAAET DVEDPEVECG(SEQ ID NO: 497), CLRDTVLLLHGLSQKDKLFMMHCVEVLHQFDQVMPGVSMLIRGLPDVTDC(SEQ ID NO: 498), and/orMLHRQWLTVRRAGGPPRTDQQRRTVRCLRDTVLLLHGLSQKDKLFMMHCVEVLHQFDQVMPGVSMLIRGLPDVTDCEEAALDDLCAAETDVEDPEVECG (SEQ ID NO: 490).Polynucleotides encoding these polypeptides are also provided.

[0626] In another embodiment, polypeptides comprising the amino acidsequence of the open reading frame upstream of the predicted signalpeptide are contemplated by the present invention. Specifically,polypeptides of the invention comprise the following amino acidsequence: GXANPEDSVCILEGFSVTALSILQHLVCHSGAVRLPITVRSGGRFCCWGRKQEPGSQXSDGDMTSALRGVADDQGQHPLLKMLLHLLAFSSAATGHLQASVLTQCLKVLVKLAENTSCDFLPRFQCVFQVLPKCLSPETPLPSVLLAVELLSLLADHDQLAPQLCSHSEGCLLLLLYMYITSRPDRVALETQWLQLEQEVVWLLAKLGV QEPLAPSHWLQLPV (SEQID NO: 494). Polynucleotides encoding these polypeptides are alsoprovided.

[0627] The gene encoding the disclosed cDNA is believed to reside onchromosome 3. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 3.

[0628] This gene is expressed primarily in breast, prostate, and to alesser extent in testes.

[0629] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, disordersaffecting the reproductive organs of both males and females, especiallycancers. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thereproductive system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., reproductive, cancerous and wounded tissues) or bodily fluids(e.g., lymph, amniotic fluid, seminal fluid, breast milk, serum, plasma,urine, synovial fluid and spinal fluid) or another tissue or cell sampletaken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.

[0630] The tissue distribution primarily in breast, prostate, and to alesser extent in testes indicates that polynucleotides and polypeptidescorresponding to this gene are useful for the diagnosis and treatment ofdisorders affecting the reproductive organs of males and females,including but not limited to cancers. Furthermore, the protein may alsobe used to determine biological activity, to raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0631] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:89 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1112 of SEQID NO:89, b is an integer of 15 to 1126, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:89, and whereb is greater than or equal to a +14.

[0632] Features of Protein Encoded by Gene No: 80

[0633] The translation product of this gene shares sequence homologywith epsilon-COP which is part of coatomers which are thought to beimportant in maintaining Golgi structure and in mediatingER-through-Golgi transport, and which can influence normal endocyticrecycling of LDL receptors (See Genbank Accession No. gi12443869(AC002985); all references available through this accession are herebyincorporated by reference herein).

[0634] Preferred polypeptides of the invention comprise the followingamino acid sequence:MSGQLDARPAAALHPQGLAHPLWTCLLPRKGPSEVPQRPPQLWVVSISVLQGQHRGRAGPRDEQSVDVTNTTFLLMAASIYLHDQNPDAALRALHQGDSLEW (SEQ ID NO: 499),SVDVTNTTFLLMAASIYLHD (SEQ ID NO: 500), QNPDAALRALHQGDSLE (SEQ ID NO:501), and/or RDSIVAELDREMSR (SEQ ID NO: 502). Polynucleotides encodingthese polypeptides are also provided. A preferred polypeptide fragmentof the invention comprises the following amino acid sequence:MLGLLLLCTPRAWLTLSGPVCFQGRDPLRSHRGHPSCGS (SEQ ID NO: 503).Polynucleotides encoding these polypeptides are also provided.

[0635] The gene encoding the disclosed cDNA is believed to reside onchromosome 19. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 19.

[0636] This gene is expressed primarily in breast tissue.

[0637] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, disordersaffecting the immune and reproductive systems, particularly of themammary glands. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune and reproductive systems, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., reproductive, cancerous and woundedtissues) or bodily fluids (e.g., breast milk, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO: 211 as residues: Gly-24 to Gln-36, Gly-47 to His-66. Polynucleotidesencoding said polypeptides are also provided.

[0638] The tissue distribution in breast tissue and homology toepsilon-COP indicates that polynucleotides and polypeptidescorresponding to this gene are useful for the diagnosis and treatment ofdisorders affecting the immune and reproductive systems, includingcancers, which arise from abnormalities in coatomer function,particularly of those tissues actively involved in secretory functions.Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0639] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:90 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1023 of SEQID NO:90, b is an integer of 15 to 1037, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:90, and whereb is greater than or equal to a +14.

[0640] Features of Protein Encoded by Gene No: 81

[0641] The translation product of this gene shares sequence homologywith the highly conserved epoxide hydrolase which is thought to have animportant function in the catalysis of potentially toxic or carcinogenicepoxides into their corresponding, inert diols (See e.g., GenbankAccession No. gi|485136; all references available through this accessionare hereby incorporated by reference herein). Preferred polypeptides ofthe invention comprise the following amino acid sequence: HGFPEFWYSWR(SEQ ID NO: 504), ASHWLQQDQP (SEQ ID NO: 505), PINHYRNIF (SEQ ID NO:506), YPEMVMKLI (SEQ ID NO: 507), PEFWYSWRYQLREF (SEQ ID NO: 508),HDWGGMIAW (SEQ ID NO: 509) Polynucleotides encoding these polypeptidesare also provided.

[0642] This gene is expressed primarily in benign and malignant prostatetissue.

[0643] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, disordersof the prostate and liver, particularly cancers. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the reproductive system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., hepatic, prostate, cancerous andwounded tissues) or bodily fluids (e.g., lymph, seminal fluid, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 212 as residues: Gln-38 toPro-49, Glu-104 to Tyr-109, His-127 to Lys-132, Thr-236 to Cys-243,Gln-328 to Asp-333, Lys-344 to Asp-351. Polynucleotides encoding saidpolypeptides are also provided.

[0644] The tissue distribution in tumors of prostate origins indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for diagnosis and intervention of these tumors, in addition toother tumors where expression has been indicated. Furthermore, theprotein may also be used to determine biological activity, raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues. Alternatively,homology to epoxide hydrolase indicates that polynucleotides andpolypeptides corresponding to this gene are useful for the detection andtreatment of liver disorders and cancers (e.g., hepatoblastoma,jaundice, hepatitis, liver metabolic diseases and conditions that areattributable to the differentiation of hepatocyte progenitor cells). Inaddition the expression in fetus would suggest a useful role for theprotein product in developmental abnormalities, fetal deficiencies,pre-natal disorders and various would-healing models and/or tissuetrauma.

[0645] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:91 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1302 of SEQID NO:91, b is an integer of 15 to 1316, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:91, and whereb is greater than or equal to a +14.

[0646] Features of Protein Encoded by Gene No: 82

[0647] This gene is expressed primarily in merkel cells.

[0648] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, disordersof the immune system. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.immune, cancerous and wounded tissues) or bodily fluids (e.g. lymph,serum, plasma, urine, synovial fluid and spinal fluid) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 213 as residues: Lys-23 toLys-29. Polynucleotides encoding said polypeptides are also provided.

[0649] The tissue distribution indicates that polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosis andtreatment of a variety of immune system disorders. Expression of thisgene product in immune tissue indicates a role in the regulation of theproliferation; survival; differentiation; and/or activation ofpotentially all hematopoietic cell lineages, including blood stem cells.This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer e.g. by boosting immuneresponses. Expression in cells of lymphoid origin, the natural geneproduct may be involved in immune functions. Therefore it may be alsoused as an agent for immunological disorders including arthritis,asthma, immune deficiency diseases such as AIDS, and leukemia. Protein,as well as, antibodies directed against the protein may show utility asa tumor marker and/or immunotherapy targets for the above listed tumorsand tissues. In addition, this gene product may have commercial utilityin the expansion of stem cells and committed progenitors of variousblood lineages, and in the differentiation and/or proliferation ofvarious cell types. Protein, as well as, antibodies directed against theprotein may show utility as a tumor marker and/or immunotherapy targetsfor the above listed tumors and tissues.

[0650] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:92 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1007 of SEQID NO:92, b is an integer of 15 to 1021, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:92, and whereb is greater than or equal to a +14.

[0651] Features of Protein Encoded by Gene No: 83

[0652] Preferred polypeptides comprise the amino acid sequence:RLGAVLTPVIPALWEAEASRSPETRSLRPAW (SEQ ID NO: 510). Polynucleotidesencoding this polypeptide are also provided.

[0653] This gene is expressed primarily in liver tissue, particularlyhepatomas.

[0654] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, disordersof the liver, including cancers. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe hepatic and hematopoietic systems, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., hepatic, cancerous and woundedtissues) or bodily fluids (e.g., lymph, bile, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO: 214 as residues: Met-i to Ser-7, His-66 to Phe-72. Polynucleotidesencoding said polypeptides are also provided.

[0655] The tissue distribution in liver indicates that polynucleotidesand polypeptides corresponding to this gene are useful for the detectionand treatment of liver disorders and cancers (e.g., hepatoblastoma,jaundice, hepatitis, liver metabolic diseases and conditions that areattributable to the differentiation of hepatocyte progenitor cells). Inaddition the expression in fetus would suggest a useful role for theprotein product in developmental abnormalities, fetal deficiencies,pre-natal disorders and various would-healing models and/or tissuetrauma. Furthermore, the protein may also be used to determinebiological activity, raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0656] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:93 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1246 of SEQID NO:93, b is an integer of 15 to 1260, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:93, and whereb is greater than or equal to a +14.

[0657] Features of Protein Encoded by Gene No: 84

[0658] Preferred polypeptides of the invention comprise the followingamino acid sequence: GSLPPKPIYLVVPR (SEQ ID NO: 511). Polynucleotidesencoding these polypeptides are also provided.

[0659] This gene is expressed primarily in skin.

[0660] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, disordersaffecting the skin, such as melanoma and wound healing, in addition toother disorders affecting the integumentary system. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system and skin, expressionof this gene at significantly higher or lower levels may be routinelydetected in certain tissues or cell types (e.g., epithelial, cancerousand wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,urine, synovial fluid and spinal fluid) or another tissue or cell sampletaken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.Preferred polypeptides of the present invention comprise immunogenicepitopes shown in SEQ ID NO: 215 as residues: Cys-56 to Pro-73, Pro-83to Lys-92. Polynucleotides encoding said polypeptides are also provided.

[0661] The tissue distribution in skin and skin melanoma indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor diagnosis and intervention of various skin disorders including skintumors, in addition to other tumors where expression has been indicated.Representative uses are described in the “Biological Activity”,“Hyperproliferative Disorders”, “Infectious Disease”, and “Regeneration”sections below, in Example 11, 19, and 20, and elsewhere herein.Briefly, the protein is useful in detecting, treating, and/or preventingcongenital disorders (i.e. nevi, moles, freckles, Mongolian spots,hemangiomas, port-wine syndrome), integumentary tumors (i.e. keratoses,Bowen's disease, basal cell carcinoma, squamous cell carcinoma,malignant melanoma, Paget's disease, mycosis fungoides, and Kaposi'ssarcoma), injuries and inflammation of the skin (i.e. wounds, rashes,prickly heat disorder, psoriasis, dermatitis), atherosclerosis,uticaria, eczema, photosensitivity, autoimmune disorders (i.e., lupuserythematosus, vitiligo, dermatomyositis, morphea, scleroderma,pemphigoid, and pemphigus), keloids, striae, erythema, petechiae,purpura, and xanthelasma. In addition, such disorders may predisposeincreased susceptibility to viral and bacterial infections of the skin(i.e., cold sores, warts, chickenpox, molluscum contagiosum, herpeszoster, boils, cellulitis, erysipelas, impetigo, tinea, althlete's foot,and ringworm).

[0662] Moreover, the protein product of this gene may also be useful forthe treatment or diagnosis of various connective tissue disorders (i.e.,arthritis, trauma, tendonitis, chrondomalacia and inflammation, etc.),autoimmune disorders (i.e., rheumatoid arthritis, lupus, scleroderma,dermatomyositis, etc.), dwarfism, spinal deformation, jointabnormalities, and chondrodysplasias (i.e. spondyloepiphyseal dysplasiacongenita, familial osteoarthritis, Atelosteogenesis type II,metaphyseal chondrodysplasia type Schmid). Furthermore, the protein mayalso be used to determine biological activity, raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0663] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:94 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 976 of SEQID NO:94, b is an integer of 15 to 990, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:94, and where bis greater than or equal to a +14.

[0664] Features of Protein Encoded by Gene No: 85

[0665] When tested against kidney K562 cell lines, supernatants removedfrom cells containing this gene activated the interferon-sensitiveresponsive element (ISRE) pathway. Thus, it is likely that this geneactivates kidney or endothelial cells through the ISRE signaltransduction pathway. ISRE is a promoter element found upstream in manygenes which are involved in the Jaks-STAT pathway. The Jaks-STAT pathwayis a large, signal transduction pathway involved in the differentiationand proliferation of cells. Therefore, activation of the Jaks-STATspathway, reflected by the binding of the ISRE element, can be used toindicate proteins involved in the proliferation and differentiation ofcells. This gene maps to chromosome 10, and therefore, may be used as amarker in linkage analysis for chromosome 10.

[0666] This gene is expressed primarily in placenta, and to a lesserextent in many other tissues or cells.

[0667] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, vasculardisease including occlusion of vessels and arteries. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the vascular system, expression ofthis gene at significantly higher or lower levels may be routinelydetected in certain tissues or cell types (e.g., reproductive, cancerousand wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid,serum, plasma, urine, synovial fluid and spinal fluid) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 216 as residues: His-58 toGly-68, Thr-76 to Arg-81. Polynucleotides encoding said polypeptides arealso provided.

[0668] The tissue distribution in placenta combined with the biologicalactivity data indicates that polynucleotides and polypeptidescorresponding to this gene are useful for the diagnosis and treatment ofcancer and other proliferative disorders. Expression within highlyvascularized tissue and other cellular sources marked by proliferatingcells indicates that this protein may play a role in the regulation ofcellular division. Additionally, the expression in placenta indicatesthat this protein may play a role in the proliferation, differentiation,and/or survival of hematopoietic cell lineages. In such an event, thisgene may be useful in the treatment of lymphoproliferative disorders,and in the maintenance and differentiation of various hematopoieticlineages from early hematopoietic stem and committed progenitor cells.Similarly, embryonic development also involves decisions involving celldifferentiation and/or apoptosis in pattern formation. Thus this proteinmay also be involved in apoptosis or tissue differentiation and couldagain be useful in cancer therapy. Furthermore, the protein may also beused to determine biological activity, to raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0669] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:95 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1696 of SEQID NO:95, b is an integer of 15 to 1710, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:95, and whereb is greater than or equal to a +14.

[0670] Features of Protein Encoded by Gene No: 86

[0671] This gene is Apolipoprotein M (See, e.g., Genbank Accession No.gb|AAD18084.1|(AF129756) and gb|AAD11443.1|(AF118393); all referencesavailable through these accessions are hereby incorporated by referenceherein). The protein components of human lipoproteins, apolipoproteins,allow the redistribution of cholesterol from the arterial wall to othertissues and exert beneficial effects on systems involved in thedevelopment of arterial lesions, like inflammation and hemostasis.

[0672] The gene encoding the disclosed cDNA is believed to reside onchromosome 6. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 6.

[0673] This gene is expressed primarily in fetal liver, fetal spleen,and to a lesser extent in adult liver, hepatocellular tumors, retina andtestis.

[0674] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,proliferative disorders of the blood and tumors of the liver ordisorders of lipid metabolism. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe immune, metabolic, and hepatic systems, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., liver, hematopoietic, cancerous andwounded tissues) or bodily fluids (e.g., bile, lymph, serum, plasma,urine, synovial fluid and spinal fluid) or another tissue or cell sampletaken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.Preferred polypeptides of the present invention comprise immunogenicepitopes shown in SEQ ID NO: 217 as residues: Glu-106 to Lys-120,Glu-136 to Tyr-141, Asn-148 to Pro-154. Polynucleotides encoding saidpolypeptides are also provided.

[0675] The tissue distribution of the gene product, ApoM, in fetalliver, and adult liver indicates that polynucleotides and polypeptidescorresponding to this gene are useful for the diagnosis, treatment andprevention of lipid metabolism disorders, including but not limited to,vascular disease, such as coronary artery disease, arteriosclerosis,and/or atherosclerosis Additionally, the tissue distribution in fetalliver and spleen indicates that polynucleotides and polypeptidescorresponding to this gene are useful for the diagnosis and treatment ofa variety of immune system disorders. Representative uses are describedin the “Immune Activity” and “Infectious Disease” sections below, inExample 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.Briefly, the expression of this gene product in fetal tissues indicatesa role in regulating the proliferation; survival; differentiation;and/or activation of potentially all hematopoietic cell lineages,including blood stem cells. This gene product may be involved in theregulation of cytokine production, antigen presentation, or otherprocesses that may also suggest a usefulness in the treatment of cancere.g., by boosting immune responses. Expression in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,and leukemia. In addition, this gene product may have commercial utilityin the expansion of stem cells and committed progenitors of variousblood lineages, and in the differentiation and/or proliferation ofvarious cell types. Furthermore, the protein may also be used todetermine biological activity, to raise antibodies, as tissue markers,to isolate cognate ligands or receptors, to identify agents thatmodulate their interactions, in addition to its use as a nutritionalsupplement. Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tissues. Alternatively, expression within liver tissuesindicates that polynucleotides and polypeptides corresponding to thisgene are useful for the detection and treatment of liver disorders andcancers (e.g. hepatoblastoma, jaundice, hepatitis, liver metabolicdiseases and conditions that are attributable to the differentiation ofhepatocyte progenitor cells). In addition the expression in fetus wouldsuggest a useful role for the protein product in developmentalabnormalities, fetal deficiencies, pre-natal disorders and variouswould-healing models and/or tissue trauma.

[0676] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:96 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 767 of SEQID NO:96, b is an integer of 15 to 781, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:96, and where bis greater than or equal to a +14.

[0677] Features of Protein Encoded by Gene No: 87

[0678] This gene is expressed primarily in LPS treated neutrophils.

[0679] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, chronicor acute inflammatory disease, and hematopoietic disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., hematopoietic, cancerous andwounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0680] The tissue distribution in neutrophils indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and diagnosis of hematopoietic related disorders suchas anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia sincestromal cells are important in the production of cells of hematopoieticlineages. Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Briefly, the uses include bone marrowcell ex-vivo culture, bone marrow transplantation, bone marrowreconstitution, radiotherapy or chemotherapy of neoplasia.

[0681] The gene product may also be involved in lymphopoiesis,therefore, it can be used in immune disorders such as infection,inflammation, allergy, immunodeficiency, etc. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Furthermore,the protein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0682] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:97 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1099 of SEQID NO:97, b is an integer of 15 to 1113, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:97, and whereb is greater than or equal to a +14.

[0683] Features of Protein Encoded by Gene No: 88

[0684] The translation product of this gene shares sequence homologywith prolylcarboxypeptidase which is thought to be important in theprocessing of bioactive peptides like angiotensin and bradykinin (SeeGenbank Accession No. gb|AAA99891.11; all references available throughthis accession are hereby incorporated by reference herein). Preferredpolypeptides comprise the following amino acid sequence:LVFAEHRYYGKSLPFG (SEQ ID NO: 512), EQALADFAEL (SEQ ID NO: 513),GGSYGGMLSAYLRMKYPH (SEQ ID NO: 514), NIIFSNGNLDPWAGGG (SEQ ID NO: 515),AMMDYPYPTDFLGPLPANPVKV (SEQ ID NO: 516), and/or FYTGNEGD (SEQ ID NO:517). Also preferred are the polynucleotides encoding thesepolypeptides. An additional preferred polypeptide fragment of theinvention comprises the following amino acid sequence:MGSAPWAPVLLLALGLRGLQAGARSGPRLPGALLPAASGPLQLRALRQQDLPSALPGVGQVLGPGRGAHLLLHWERGRRVGLRQQLGLRRGLAAERGALLVFAEHRYYGKSLPFGAQSTQRGHTELLTVEQALADFAELLRALRRDLGAQDAPAIAFGGSYGGMLSAYLRMKYPHLVAGALAASAPVLSVAGLGDSNQFFRDVTADFEGQSPKCTQGVREAFRQIKDLFLQGAYDTVRWEFGTCQPLSDEKDLTQLFMFARNAFTVLAMMDYPYPTDFLGPLPANPVKVGCDRLLSEAQRITGLRALAGLVYNASGSEHCYDIYRLYHSCADPTGCGTGPDARAWDYQACTEINLTFASNNVTDMFPDLPFTDELRQRYCLDTWGVWPRPDWLLTSFWGGDLRAASNIIFSNGNLDPWAGGGIRRNLSASVIAVTIQGGAHHLDLRASHPEDPASVVEARKLEATII GEWVKAARREQQPALRGGPRLSL (SEQ ID NO: 518). Polynucleotides encoding thesepolypeptides are also provided.

[0685] This gene is expressed primarily in uterine cancer, testis, andto a lesser extent in lymph nodes, dendritic cells and HL60 cell line.

[0686] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, uterinecancer, reproductive, and immune disorders. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the reproductive system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., reproductive, cancerous and woundedtissues) or bodily fluids (e.g., amniotic fluid, seminal fluid, lymph,serum, plasma, urine, synovial fluid and spinal fluid) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 219 as residues: Gly-23 toAla-30, Pro-44 to Phe-54, Glu-69 to Pro-77, Gln-142 to His-148, Phe-232to Gly-242, Pro-271 to Leu-278, Ser-340 to Asp-347, Pro-365 to Asp-371,Asp-398 to Leu-406, Arg-500 to Pro-505. Polynucleotides encoding saidpolypeptides are also provided.

[0687] The tissue distribution in uterine cancer and homology toprolylcarboxypeptidase indicates that the protein product of this genewould is useful for diagnosis, treatment and prevention of diseasesassociated with the reproductive system including uterine cancer, aswell as, cardiovascular diseases where prolylcarboxypeptidases primarysubstrate, angiotension, has its greatest affect. In addition, theputative location of prolylcarboxypeptidase within the lysosomalcompartment of cells indicates that polynucleotides and polypeptidescorresponding to this gene are useful for the diagnosis, prevention,and/or treatment of various metabolic disorders such as Tay-Sachsdisease, phenylkenonuria, galactosemia, porphyrias, and Hurler'ssyndrome. Furthermore, the protein may also be used to determinebiological activity, to raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0688] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:98 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1709 of SEQID NO:98, b is an integer of 15 to 1723, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:98, and whereb is greater than or equal to a +14.

[0689] Features of Protein Encoded by Gene No: 89

[0690] The translation product of this gene shares sequence homologywith the human CGI-06 protein (See, e.g. Genbank Accession No.gb|AAD27715.1|AF132940_(—)1 (AF132940); all references available throughthis accession are hereby incorporated by reference herein).

[0691] When tested against the myeloid cell line, U937, supernatantsremoved from cells containing this gene activated the GAS (gammaactivation site) pathway. Thus, it is likely that this gene activatesmyeloid cells through the Jaks-STAT signal transduction pathway. The GAS(gamma activation site) is a promoter element found upstream in manygenes which are involved in the Jaks-STAT pathway. The Jaks-STAT pathwayis a large, signal transduction pathway involved in the differentiationand proliferation of cells. Therefore, activation of the Jaks-STATspathway, reflected by the binding of the GAS element, can be used toindicate proteins involved in the proliferation and differentiation ofcells.

[0692] The gene encoding the disclosed cDNA is believed to reside onchromosome 20. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 20.

[0693] This gene is expressed primarily in various tumors includingendometrial tumors, adenocarcinoma, breast cancer, osteosarcoma,chondrosarcoma, uterine and pancreas tumors and to a lesser extent inembryonic tissues.

[0694] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,identification and treatment of many types of solid tumors. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the major organs, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., skeletal, reproductive,cancerous and wounded tissues) or bodily fluids (e.g., breast milk,lymph, serum, plasma, urine, synovial fluid and spinal fluid) or anothertissue or cell sample taken from an individual having such a disorder,relative to the standard gene expression level, i.e., the expressionlevel in healthy tissue or bodily fluid from an individual not havingthe disorder. Preferred polypeptides of the present invention compriseimmunogenic epitopes shown in SEQ ID NO: 220 as residues: Pro-25 toArg-31, Thr-52 to Val-63, Asn-129 to Lys-135, Gln-197 to Trp-202,Thr-230 to Glu-236, Pro-242 to Tyr-248, Leu-280 to Pro-291, Ser-348 toSer-356, Pro-362 to Gln-368, Thr-398 to His-406, Trp-430 to Leu-435,Glu-499 to Gly-504. Polynucleotides encoding said polypeptides are alsoprovided.

[0695] The tissue distribution in solid tumors combined with theGAS-element activity indicates that polynucleotides and polypeptidescorresponding to this gene are useful for the diagnosis and treatment ofcancer and other proliferative disorders. Expression within embryonictissue and other cellular sources marked by proliferating cellsindicates that this protein may play a role in the regulation ofcellular division. Representative uses are described in the“Hyperproliferative Disorders” and “Regeneration” sections below andelsewhere herein. Briefly, developmental tissues rely on decisionsinvolving cell differentiation and/or apoptosis in pattern formation.Dysregulation of apoptosis can result in inappropriate suppression ofcell death, as occurs in the development of some cancers, or in failureto control the extent of cell death, as is believed to occur in acquiredimmunodeficiency and certain neurodegenerative disorders, such as spinalmuscular atrophy (SMA). Because of potential roles in proliferation anddifferentiation, this gene product may have applications in the adultfor tissue regeneration and the treatment of cancers. It may also act asa morphogen to control cell and tissue type specification. Therefore,the polynucleotides and polypeptides of the present invention are usefulin treating, detecting, and/or preventing said disorders and conditions,in addition to other types of degenerative conditions. Thus this proteinmay modulate apoptosis or tissue differentiation and is useful in thedetection, treatment, and/or prevention of degenerative or proliferativeconditions and diseases. The protein is useful in modulating the immuneresponse to aberrant polypeptides, as may exist in proliferating andcancerous cells and tissues. The protein can also be used to gain newinsight into the regulation of cellular growth and proliferation.Additionally, the expression in hematopoietic cells and tissuesindicates that this protein may play a role in the proliferation,differentiation, and/or survival of hematopoietic cell lineages. In suchan event, this gene may be useful in the treatment oflymphoproliferative disorders, and in the maintenance anddifferentiation of various hematopoietic lineages from earlyhematopoietic stem and committed progenitor cells. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0696] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:99 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 2073 of SEQID NO:99, b is an integer of 15 to 2087, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:99, and whereb is greater than or equal to a +14.

[0697] Features of Protein Encoded by Gene No: 90

[0698] This gene is expressed primarily in brain medulloblastoma cells.

[0699] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis of brainmedulloblastoma and other neurological disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the central nervous system, expressionof this gene at significantly higher or lower levels may be routinelydetected in certain tissues or cell types (e.g., neural, cancerous andwounded issues) or bodily fluids (e.g., lymph, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0700] The tissue distribution in medulloblastoma indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the detection, treatment, and/or prevention of neurodegenerativedisease states, behavioral disorders, or inflammatory conditionsRepresentative uses are described in the “Regeneration” and“Hyperproliferative Disorders” sections below, in Example 11, 15, and18, and elsewhere herein. Briefly, the uses include, but are not limitedto the detection, treatment, and/or prevention of Alzheimer's Disease,Parkinson's Disease, Huntington's Disease, Tourette Syndrome,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,panic disorder, learning disabilities, ALS, psychoses, autism, andaltered behaviors, including disorders in feeding, sleep patterns,balance, and perception. In addition, the gene or gene product may alsoplay a role in the treatment and/or detection of developmental disordersassociated with the developing embryo or disorders of the cardiovascularsystem. Protein, as well as, antibodies directed against the protein mayshow utility as a tumor marker and/or immunotherapy targets for theabove listed tumors and tissues.

[0701] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:100 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 737 of SEQID NO:100, b is an integer of 15 to 751, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:100, andwhere b is greater than or equal to a +14.

[0702] Features of Protein Encoded by Gene No: 91

[0703] This gene maps to the chromosome X, and therefore, may be used asa marker in linkage analysis for chromosome X.

[0704] Preferred polypeptides of the invention comprise the followingamino acid sequence: CSVFPPSLWFYLPLVFDDGDVQ (SEQ ID NO: 519),GVSLPLLGDASQLGYLGVRDALEEALCLFSDVQLCAGRTSALFKAXRQGRLSLQRILLPFVWLCPAPQRWSLQRQAGLLELRWAPPSSSFLAALFTPSSLGNGGR PSPSLTAXL QFDLRLLC(SEQ ID NO: 520), and/orVCRGFCCLLFGCALPPRGGVYRGRQASLNCGGLHRVRVSWPLCLPPQASAM VGAPPPASLPXCSLISDCCASNX SEQ ID NO: 521(SEQ ID NO: 491). Polynucleotides encodingthese polypeptides are also provided.

[0705] This gene is expressed primarily in spleen from chroniclymphocytic leukemia patients.

[0706] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, chroniclymphocytic leukemia, and other immune disorders, particularlyproliferative diseases. Similarly, polypeptides and antibodies directedto these polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph,serum, plasma, urine, synovial fluid and spinal fluid) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

[0707] The tissue distribution in spleen from chronic lymphocyticleukemia patients indicates that polynucleotides and polypeptidescorresponding to this gene are useful for the diagnosis and treatment ofa variety of immune system disorders. Representative uses are describedin the “Immune Activity” and “Infectious Disease” sections below, inExample 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.Briefly, the expression of this gene product in leukemia cells indicatesa role in the regulation of the proliferation; survival;differentiation; and/or activation of potentially all hematopoietic celllineages, including blood stem cells. This gene product may be involvedin the regulation of cytokine production, antigen presentation, or otherprocesses that may also suggest a usefulness in the treatment of cancere.g., by boosting immune responses. Expression in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,and leukemia. In addition, this gene product may have commercial utilityin the expansion of stem cells and committed progenitors of variousblood lineages, and in the differentiation and/or proliferation ofvarious cell types. Furthermore, the protein may also be used todetermine biological activity, raise antibodies, as tissue markers, toisolate cognate ligands or receptors, to identify agents that modulatetheir interactions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0708] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:101 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1209 of SEQID NO:101, b is an integer of 15 to 1223, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:101, andwhere b is greater than or equal to a +14.

[0709] Features of Protein Encoded by Gene No: 92

[0710] The translation product of this gene was shown to have homologyto the human reverse transcriptase gene (See e.g., Genbank Accession No.gi|439877; all references available through this accession are herebyincorporated by reference herein).

[0711] Preferred polypeptides of the invention comprise the followingamino acid sequence: MS RRSATSYIIRERQIKIIVRYHYTPIMT (SEQ ID NO: 522),IRERQIKIIVRYHYTP (SEQ ID NO: 523), KKTCTMFIATLFI (SEQ ID NO: 524),SVASVFIPLKVSVTKQFIFFXFFFFLRRSLAPAWVAERXTSQETKQNKKTPQLRGKVAHACDPITLGGRRWEVGESLEARSPS (SEQ ID NO: 526) and/or EKIFAKHLSVKGL (SEQID NO: 525). Polynucleotides encoding these polypeptides are alsoprovided.

[0712] The gene encoding the disclosed cDNA is believed to reside onchromosome 22. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 22.

[0713] This gene is expressed primarily in microvascular endothelialcells.

[0714] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, variousdiseases of the cardiovascular and circulatory systems. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the cardiovascular system, expressionof this gene at significantly higher or lower levels may be routinelydetected in certain tissues or cell types (e.g., vascular, cancerous andwounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0715] The tissue distribution in microvascular endothelial cellscombined with the homology to the conserved human gene for reversetranscriptase indicates that polynucleotides and polypeptidescorresponding to this gene are useful for the diagnosis and treatment ofcancer and other proliferative disorders, particularly vasculardisorders. Representative uses are described in the “Immune Activity”and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18,19, 20, and 27, and elsewhere herein. Alternatively expression withinmicrovascular tissue, a tissue marked by proliferating cells, indicatesthat this protein may play a role in the regulation of cellulardivision. As such, this protein may play a role in the proliferation,differentiation, and/or survival of hematopoietic cell lineages. In suchan event, this gene may be useful in the treatment oflymphoproliferative disorders, and in the maintenance anddifferentiation of various hematopoietic lineages from earlyhematopoietic stem and committed progenitor cells. Similarly, embryonicdevelopment also involves decisions involving cell differentiationand/or apoptosis in pattern formation. Thus this protein may also beinvolved in apoptosis or tissue differentiation and could again beuseful in cancer therapy. Furthermore, the protein may also be used todetermine biological activity, to raise antibodies, as tissue markers,to isolate cognate ligands or receptors, to identify agents thatmodulate their interactions, in addition to its use as a nutritionalsupplement. Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tissues.

[0716] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:102 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 996 of SEQID NO:102, b is an integer of 15 to 1010, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:102, andwhere b is greater than or equal to a +14.

[0717] Features of Protein Encoded by Gene No: 93

[0718] The translation product of this gene shares sequence homologywith the Y43F4B.5 protein from Caenorhabditis elegans (See GenbankAccession No. gnl|PID|e1247424 (AL021481)).

[0719] Moreover, the translation product also shares homology tophosphoglucomutase and phosphomannomutase proteins (See GenbankAccessions CAA16334.1 and CAA20128.1; all references available throughthis accession are hereby incorporated herein by reference). Based onthe sequence similarity, the translation product of this gene isexpected to share at least some biological activities withphosphoglucomutase proteins. Such activities are known in the art, someof which are described elsewhere herein.

[0720] Preferred polypeptides of the invention comprise the followingamino acid sequence:ARGKTVLFAFEEAIGYMCCPFVLDKDGVSAAVISAELASFLATKNLSLSQQLKAIYVEYGYHITKASYFICHDQETIKKLFENLRNYDGKNNYPKACGKFEISAIRDLTTGYDDSQPDKKAVLPTSKSSQMITFTFANGGVATMRTSGTEPKIKYYAELCAPPGNSDPEQLKKELNELVSAIEEHFFQPQKYNLQPKAD (SEQ ID NO: 528),YMCCPFVLDKDGVSAAVISAELASFLATKNLSLSQQLKAIYVEYGYHITKASYFICHDQETIKKLFENLRNYDGKNNYPKACGKFEISAIRDLTTGYDDSQPDKKAVLPTSKSSQMITFTFANGGVATMRTSGTEPKIKYYAELCAPPGNSDPEQLKKELNELVSAIEEHFFQPQKYNLQPKAD (SEQ ID NO: 527), DKDGVSAAVISAELASFL (SEQ IDNO: 529), RDLTTGYDDSQPD (SEQ ID NO: 530), KAVLPTSKSSQMITF (SEQ ID NO:531), and/or TMRTSGTEPKIKYYAEL (SEQ ID NO: 532). Polynucleotidesencoding these polypeptides are also provided.

[0721] The gene encoding the disclosed cDNA is believed to reside onchromosome 4. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 4.

[0722] This gene is expressed primarily in placenta, fetal spleen, andto a lesser extent in prostate, T-cells and neutophils.

[0723] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, variousdiseases of the immune and reproductive systems, including cancer.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the immune andreproductive systems, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., immune, reproductive, cancerous and wounded tissues) or bodilyfluids (e.g., seminal fluid, lymph, serum, plasma, urine, synovial fluidand spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder. Preferred polypeptidesof the present invention comprise immunogenic epitopes shown in SEQ IDNO: 224 as residues: Leu-23 to Met-30. Polynucleotides encoding saidpolypeptides are also provided.

[0724] The tissue distribution in fetal spleen indicates polynucleotidesand polypeptides corresponding to this gene are useful for the diagnosisand treatment of a variety of immune system disorders. Representativeuses are described in the “Immune Activity” and “Infectious Disease”sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, andelsewhere herein. Briefly, the expression of this gene product indicatesa role in regulating the proliferation; survival; differentiation;and/or activation of hematopoietic cell lineages, including blood stemcells. Involvement in the regulation of cytokine production, antigenpresentation, or other processes suggests a usefulness in the treatmentof cancer (e.g., by boosting immune responses). Expression in cells oflymphoid origin, the natural gene product would be involved in immunefunctions. Therefore it is also useful as an agent for immunologicaldisorders including arthritis, asthma, immunodeficiency diseases such asAIDS, leukemia, rheumatoid arthritis, granulomatous disease,inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia,psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity;immune reactions to transplanted organs and tissues, such ashost-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.

[0725] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. Thus, this gene productis thought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types.

[0726] Moreover, the protein is useful in the detection, treatment,and/or prevention of a variety of vascular disorders and conditions,which include, but are not limited to miscrovascular disease, vascularleak syndrome, aneurysm, stroke, embolism, thrombosis, coronary arterydisease, arteriosclerosis, and/or atherosclerosis. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0727] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:103 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1972 of SEQID NO:103, b is an integer of 15 to 1986, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:103, andwhere b is greater than or equal to a +14.

[0728] Features of Protein Encoded by Gene No: 94

[0729] This gene is expressed primarily in activated monocytes.

[0730] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, variousdiseases and/or disorders of the immune system. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., immune, cancerous and woundedtissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0731] The tissue distribution in activated monocytes indicatespolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein. Briefly, the expression of this geneproduct indicates a role in regulating the proliferation; survival;differentiation; and/or activation of hematopoietic cell lineages,including blood stem cells. Involvement in the regulation of cytokineproduction, antigen presentation, or other processes suggests ausefulness in the treatment of cancer (e.g. by boosting immuneresponses). Expression in cells of lymphoid origin, the natural geneproduct would be involved in immune functions. Therefore it is alsouseful as an agent for immunological disorders including arthritis,asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoidarthritis, granulomatous disease, inflammatory bowel disease, sepsis,acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such asT-cell mediated cytotoxicity; immune reactions to transplanted organsand tissues, such as host-versus-graft and graft-versus-host diseases,or autoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, andscleroderma.

[0732] Moreover, the protein may represent a secreted factor thatinfluences the differentiation or behavior of other blood cells, or thatrecruits hematopoietic cells to sites of injury. Thus, this gene productis thought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0733] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:104 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1307 of SEQID NO:104, b is an integer of 15 to 1321, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:104, andwhere b is greater than or equal to a +14. TABLE 1 5′ NT of First LastNT 5′ NT 3′ NT 5′ NT First AA AA AA First Last ATCC SEQ Total of of ofAA of SEQ of of AA of AA Gene cDNA Deposit ID NT Clone Clone StartSignal ID Sig Sig Secreted of No. Clone ID NO: Z and Date Vector NO: XSeq. Seq. Seq. Codon Pep NO: Y Pep Pep Portion ORF 1 HWBBP10 209782pCMVSport 11 899 1 899 66 66 132 1 26 27 56 Apr. 20, 1998 3.0 1 HWBBP10209782 pCMVSport 105 944 1 944 55 55 226 1 26 27 56 Apr. 20, 1998 3.0 2HWBDO80 209782 pCMVSport 12 1140 1 1140 166 166 133 1 22 23 41 Apr. 20,1998 3.0 3 HWHGU54 209782 pCMVSport 13 1445 1 1445 145 145 134 1 19 20414 Apr. 20, 1998 3.0 4 HYACI76 209782 pCMVSport 14 1208 1 1148 385 385135 1 25 26 44 Apr. 20, 1998 3.0 5 HBHMA23 209782 pSport1 15 1175 2 117571 71 136 1 24 25 197 Apr. 20, 1998 5 HBHMA23 209782 pSport1 106 1172 11172 70 70 227 1 24 25 76 Apr. 20, 1998 6 HCE3G20 209782 Uni-ZAP XR 162374 1 2350 57 57 137 1 42 43 45 Apr. 20, 1998 7 HCEJP80 209782 Uni-ZAPXR 17 1595 1 1595 90 90 138 1 21 22 40 Apr. 20, 1998 8 HCUDD24 209782ZAP Express 18 1287 89 1287 314 314 139 1 19 20 84 Apr. 20, 1998 9HDPTD15 209782 pCMVSport 19 1396 1 1396 223 223 140 1 18 19 200 Apr. 20,1998 3.0 10 HDPWU34 209782 pCMVSport 20 1277 860 1277 117 117 141 1 2324 325 Apr. 20, 1998 3.0 10 HDPWU34 209782 pCMVSport 107 427 1 427 111111 228 1 16 17 44 Apr. 20, 1998 3.0 11 HEOOV79 209782 pSport1 21 1781 11767 203 203 142 1 23 24 118 Apr. 20, 1998 12 HFKET93 209782 Uni-ZAP XR22 1491 1 1491 75 75 143 1 15 16 47 Apr. 20, 1998 13 HFTDL56 209782Uni-ZAP XR 23 1839 32 1838 93 93 144 1 22 23 519 Apr. 20, 1998 14HFXJX44 209782 Lambda ZAP 24 1384 1 1384 98 98 145 1 18 19 47 Apr. 20,1998 II 15 HKACU58 209782 pCMVSport 25 1681 1 1681 98 98 146 1 18 19 431Apr. 20, 1998 2.0 15 HKACU58 209782 pCMVSport 108 1708 69 1708 117 117229 1 18 19 101 Apr. 20, 1998 2.0 16 HKFBC53 209782 ZAP Express 26 19491 1906 41 41 147 1 18 19 442 Apr. 20, 1998 16 HLDBQ19 209072 pCMVSport109 1487 401 1487 534 534 230 1 22 23 132 May 22, 1997 3.0 16 HLDBQ19209072 pCMVSport 110 1525 401 1480 534 534 231 1 22 23 66 May 22, 19973.0 17 HLTHR66 209782 Uni-ZAP XR 27 2286 1 2286 5 5 148 1 34 35 75 Apr.20, 1998 18 HLYBA69 209782 pSport1 28 530 1 530 89 89 149 1 29 30 51Apr. 20, 1998 19 HNTMX29 209782 pSport1 29 1296 756 1291 118 118 150 131 32 209 Apr. 20, 1998 19 HNTMX29 209782 pSport1 111 552 1 552 18 18232 1 18 19 72 Apr. 20, 1998 20 HNTNC20 209782 pSport1 30 1979 1 1979270 270 151 1 19 20 218 Apr. 20, 1998 21 HNTNI01 209782 pSport1 31 12741 1114 306 306 152 1 33 34 49 Apr. 20, 1998 22 HOHCK70 209782 pCMVSport32 1531 1 1531 245 245 153 1 27 28 40 Apr. 20, 1998 2.0 23 HSMBE69209782 pSport1 33 2090 1 2090 69 69 154 1 18 19 107 Apr. 20, 1998 24HT4FW61 209782 Uni-ZAP XR 34 1006 31 1006 107 107 155 1 38 39 156 Apr.20, 1998 25 HYABK95 209782 pCMVSport 35 1787 1 1787 267 267 156 1 26 27150 Apr. 20, 1998 3.0 26 HYACE88 209782 pCMVSport 36 1201 1 1180 316 316157 1 16 17 70 Apr. 20, 1998 3.0 27 HOABR60 209782 Uni-ZAP XR 37 1896 1903 45 45 158 1 16 17 490 Apr. 20, 1998 27 HOABR60 209782 Uni-ZAP XR 112925 1 903 45 45 233 1 16 17 293 Apr. 20, 1998 28 HAGCT73 209782 Uni-ZAPXR 38 1152 1 1152 119 119 159 1 30 31 31 Apr. 20, 1998 29 HAPOM45 209782Uni-ZAP XR 39 1017 34 1017 98 98 160 1 31 32 115 Apr. 20, 1998 30HCEJQ69 209782 Uni-ZAP XR 40 1777 1 1777 39 39 161 1 26 27 380 Apr. 20,1998 31 HAGFI62 209782 Uni-ZAP XR 41 1003 368 992 429 429 162 1 28 29 91Apr. 20, 1998 32 HAGGS43 209782 Uni-ZAP XR 42 1201 1 1201 62 62 163 1 2526 44 Apr. 20, 1998 33 HBJHP03 209852 Uni-ZAP XR 43 1176 1 1176 185 185164 1 20 21 45 May 07, 1998 34 HCHPF68 209852 pSport1 44 569 1 569 186186 165 1 36 37 128 May 07, 1998 35 HDPJF37 209852 pCMVSport 45 986 1986 196 196 166 1 23 24 57 May 07, 1998 3.0 36 HSDEZ20 209852 Uni-ZAP XR46 1540 1 1540 66 66 167 1 41 42 97 May 07, 1998 37 HTEKU58 209852Uni-ZAP XR 47 792 73 792 93 93 168 1 30 31 59 May 07, 1998 38 HLTBL58209852 Uni-ZAP XR 48 1497 1 1497 26 26 169 1 20 21 42 May 07, 1998 39HPWDJ42 209852 Uni-ZAP XR 49 1340 1 1340 149 149 170 1 18 19 54 May 07,1998 39 HPWDJ42 209852 Uni-ZAP XR 113 1340 1 1340 149 149 234 1 21 22 54May 07, 1998 39 HPWDJ42 209852 Uni-ZAP XR 114 813 1 813 161 161 235 1 1819 47 May 07, 1998 40 HRACD15 209852 pCMVSport 50 1539 24 1539 252 252171 1 40 41 53 May 07, 1998 3.0 40 HRACD15 209852 pCMVSport 115 1681 241453 252 252 236 1 40 41 53 May 07, 1998 3.0 41 HSIAC80 209852 Uni-ZAPXR 51 1423 1 1423 178 178 172 1 17 18 53 May 07, 1998 42 HAGFD18 209852Uni-ZAP XR 52 1364 94 1364 261 261 173 1 21 22 48 May 07, 1998 43HMTAT59 209852 pCMVSport 53 2288 501 2276 301 301 174 1 14 15 224 May07, 1998 3.0 44 HDTGC86 209852 pCMVSport 54 1512 1 1512 351 351 175 1 2728 200 May 07, 1998 2.0 45 HAGDI35 209852 Uni-ZAP XR 55 1357 1 1338 318318 176 1 25 26 93 May 07, 1998 46 HELHN47 209852 Uni-ZAP XR 56 1989 8831989 778 778 177 1 30 31 404 May 07, 1998 47 HPRBC80 209852 Uni-ZAP XR57 2543 1245 2543 94 94 178 1 30 31 387 May 07, 1998 47 HPRBC80 209852Uni-ZAP XR 116 2052 275 2032 404 404 237 1 26 27 69 May 07, 1998 48HAQAR23 209852 Uni-ZAP XR 58 777 66 777 92 92 179 1 19 20 145 May 07,1998 49 HAIFL18 209852 Uni-ZAP XR 59 879 1 879 274 274 180 1 29 30 140May 07, 1998 50 HJPAY76 209852 Uni-ZAP XR 60 1161 1 1161 134 134 181 121 22 127 May 07, 1998 51 HUSXE77 209852 pSport1 61 687 1 687 156 156182 1 20 21 146 May 07, 1998 52 HUFEF62 209852 pSport1 62 518 1 518 190190 183 1 28 29 68 May 07, 1998 52 HUFEF62 209852 pSport1 117 539 1 539182 182 238 1 28 29 68 May 07, 1998 53 HTWJK32 209852 Lambda ZAP 63 911211 911 376 376 184 1 20 21 51 May 07, 1998 II 54 HTWDF76 209852 pSport164 963 1 963 316 316 185 1 24 25 85 May 07, 1998 55 HTPBN68 209852Uni-ZAP XR 65 1001 1 1001 429 429 186 1 20 21 191 May 07, 1998 56HTOIY21 209852 Uni-ZAP XR 66 1558 1 1558 91 91 187 1 14 15 231 May 07,1998 57 HTLDD53 209852 Uni-ZAP XR 67 1322 1 1322 162 162 188 1 25 26 68May 07, 1998 58 HTLFG05 209852 Uni-ZAP XR 68 865 1 717 137 137 189 1 3031 211 May 07, 1998 58 HTLFG05 209852 Uni-ZAP XR 118 882 1 882 137 137239 1 30 31 67 May 07, 1998 59 HDPXR23 209852 pCMVSport 69 1150 20 115049 49 190 1 20 21 90 May 07, 1998 3.0 59 HDPXR23 209852 pCMVSport 1191193 1 1189 95 95 240 1 20 21 90 May 07, 1998 3.0 60 HSIAC45 209852Uni-ZAP XR 70 1398 1 1398 12 12 191 1 23 24 62 May 07, 1998 61 HSRGW16209853 Uni-ZAP XR 71 1557 180 1007 72 72 192 1 12 13 295 May 07, 1998 61HSRGW16 209853 Uni-ZAP XR 120 1338 1 1338 170 170 241 1 47 48 140 May07, 1998 62 HSSJC35 209853 Uni-ZAP XR 72 1163 1 1163 55 55 193 1 30 31295 May 07, 1998 62 HSSJC35 209853 Uni-ZAP XR 121 1183 1 1183 66 66 2421 30 31 37 May 07, 1998 63 HTEAX23 209853 Uni-ZAP XR 73 1486 1 1486 7272 194 1 20 21 338 May 07, 1998 64 HTGCH22 209853 Uni-ZAP XR 74 1553 11553 12 12 195 1 29 30 78 May 07, 1998 65 HTJMA95 209853 pCMVSport 751650 198 1569 527 527 196 1 22 23 181 May 07, 1998 2.0 66 HHEAA08 209853pCMVSport 76 2150 1 2150 88 88 197 1 38 39 79 May 07, 1998 3.0 66HHEAA08 209853 pCMVSport 122 615 1 615 311 243 1 20 May 07, 1998 3.0 67HBQAA49 209853 Lambda ZAP 77 1592 1 1592 197 197 198 1 37 38 69 May 07,1998 II 68 HDPBI32 209853 pCMVSport 78 1579 598 1184 103 103 199 1 30 31271 May 07, 1998 3.0 68 HDPBI32 209853 pCMVSport 123 587 1 587 51 51 2441 35 36 138 May 07, 1998 3.0 69 HBIBF16 209853 Uni-ZAP XR 79 1396 1 139615 15 200 1 35 36 51 May 07, 1998 70 HBCAY05 209853 Uni-ZAP XR 80 1230576 1209 627 627 201 1 22 23 71 May 07, 1998 71 HCUCK44 209853 ZAPExpress 81 1139 573 1133 593 593 202 1 30 31 60 May 07, 1998 72 HCE2W56209853 Uni-ZAP XR 82 1409 1 1409 61 61 203 1 21 22 143 May 07, 1998 73HCWAG01 209853 ZAP Express 83 714 1 714 192 192 204 1 25 26 148 May 07,1998 74 HLDBY02 209853 pCMVSport 84 1097 1 1097 326 326 205 1 30 31 36May 07, 1998 3.0 75 HDRMI82 209853 pSport1 85 1931 540 1900 170 170 2061 25 26 406 May 07, 1998 75 HDRMI82 209853 pSport1 124 1379 1 1357 328328 245 1 30 31 175 May 07, 1998 76 HEPCU48 209853 Uni-ZAP XR 86 1092 11092 98 98 207 1 26 27 91 May 07, 1998 77 HDPRK33 209853 pCMVSport 87578 1 573 99 99 208 1 44 45 101 May 07, 1998 3.0 77 HDPRK33 209853pCMVSport 125 583 1 583 109 109 246 1 21 22 101 May 07, 1998 3.0 78HKGAX42 209853 pSport1 88 699 1 699 69 69 209 1 18 19 50 May 07, 1998 79HLMAZ95 209853 Uni-ZAP XR 89 1126 7 1126 187 187 210 1 33 34 161 May 07,1998 80 HLMFC07 209853 Lambda ZAP 90 1037 1 1037 203 203 211 1 17 18 227May 07, 1998 II 80 HLMFC07 209853 Lambda ZAP 126 1268 1 1268 203 203 2471 30 31 39 May 07, 1998 II 81 HL2AG87 209853 Uni-ZAP XR 91 1316 1 1316110 110 212 1 37 38 351 May 07, 1998 82 HKGCO27 209853 pSport1 92 1021 11021 313 313 213 1 26 27 93 May 07, 1998 82 HKGCO27 209853 pSport1 1271311 1 1311 57 57 248 1 26 27 47 May 07, 1998 83 HLDCE79 209853pCMVSport 93 1260 1 1260 342 342 214 1 63 64 101 May 07, 1998 3.0 83HLDCE79 209853 pCMVSport 128 1249 1 1249 298 298 249 1 30 31 34 May 07,1998 3.0 84 HERAD40 209853 Uni-ZAP XR 94 990 1 990 85 85 215 1 38 39 98May 07, 1998 85 HFOXB55 209853 pSport1 95 1710 1 1710 138 138 216 1 3435 81 May 07, 1998 86 HFVGZ42 209853 pBluescript 96 781 1 781 71 71 2171 22 23 188 May 07, 1998 87 HNHAF39 209853 Uni-ZAP XR 97 1113 1 1113 332332 218 1 30 31 44 May 07, 1998 88 HNTSW57 209853 pSport1 98 1723 1811723 19 19 219 1 21 22 515 May 07, 1998 88 HNTSW57 209853 pSport1 1291660 1 1660 38 38 250 1 21 22 490 May 07, 1998 89 HOGCK20 209853pCMVSport 99 2087 1 2087 57 57 220 1 23 24 522 May 07, 1998 2.0 89HOGCK20 209853 pCMVSport 130 2075 1 2054 53 251 1 22 23 554 May 07, 19982.0 90 HMDAL49 209853 Uni-ZAP XR 100 751 1 751 52 52 221 1 22 23 52 May07, 1998 91 HLYES38 209853 pSport1 101 1223 1 1223 69 69 222 1 22 23 73May 07, 1998 92 HMECK83 209853 Lambda ZAP 102 1010 1 1010 50 50 223 1 2829 54 May 07, 1998 II 93 HSHAX21 209853 Uni-ZAP XR 103 1986 1 1986 177177 224 1 13 14 72 May 07, 1998 94 HMQAG66 209853 Uni-ZAP XR 104 1321 11321 652 652 225 1 30 31 66 May 07, 1998 94 HMQAG66 209853 Uni-ZAP XR131 1333 1 1333 657 657 252 1 24 25 69 May 07, 1998

[0734] Table 1 summarizes the information corresponding to each “GeneNo.” described above. The nucleotide sequence identified as “NT SEQ IDNO:X” was assembled from partially homologous (“overlapping”) sequencesobtained from the “cDNA clone ID” identified in Table 1 and, in somecases, from additional related DNA clones. The overlapping sequenceswere assembled into a single contiguous sequence of high redundancy(usually three to five overlapping sequences at each nucleotideposition), resulting in a final sequence identified as SEQ ID NO:X.

[0735] The cDNA Clone ID was deposited on the date and given thecorresponding deposit number listed in “ATCC Deposit No:Z and Date.”Some of the deposits contain multiple different clones corresponding tothe same gene. “Vector” refers to the type of vector contained in thecDNA Clone ID.

[0736] “Total NT Seq.” refers to the total number of nucleotides in thecontig identified by “Gene No.” The deposited clone may contain all ormost of these sequences, reflected by the nucleotide position indicatedas “5′ NT of Clone Seq.” and the “3′ NT of Clone Seq.” of SEQ ID NO:X.The nucleotide position of SEQ ID NO:X of the putative start codon(methionine) is identified as “5′ NT of Start Codon.” Similarly, thenucleotide position of SEQ ID NO:X of the predicted signal sequence isidentified as “5′ NT of First AA of Signal Pep.”

[0737] The translated amino acid sequence, beginning with themethionine, is identified as “AA SEQ ID NO:Y,” although other readingframes can also be easily translated using known molecular biologytechniques. The polypeptides produced by these alternative open readingframes are specifically contemplated by the present invention.

[0738] The first and last amino acid position of SEQ ID NO:Y of thepredicted signal peptide is identified as “First AA of Sig Pep” and“Last AA of Sig Pep.” The predicted first amino acid position of SEQ IDNO:Y of the secreted portion is identified as “Predicted First AA ofSecreted Portion.” Finally, the amino acid position of SEQ ID NO:Y ofthe last amino acid in the open reading frame is identified as “Last AAof ORF.”

[0739] SEQ ID NO:X (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:Y (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further below. For instance,SEQ ID NO:X is useful for designing nucleic acid hybridization probesthat will detect nucleic acid sequences contained in SEQ ID NO:X or thecDNA contained in the deposited clone. These probes will also hybridizeto nucleic acid molecules in biological samples, thereby enabling avariety of forensic and diagnostic methods of the invention. Similarly,polypeptides identified from SEQ ID NO:Y may be used, for example, togenerate antibodies which bind specifically to proteins containing thepolypeptides and the secreted proteins encoded by the cDNA clonesidentified in Table 1.

[0740] Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

[0741] Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:X and the predicted translated amino acid sequence identified as SEQID NO:Y, but also a sample of plasmid DNA containing a human cDNA of theinvention deposited with the ATCC, as set forth in Table 1. Thenucleotide sequence of each deposited clone can readily be determined bysequencing the deposited clone in accordance with known methods. Thepredicted amino acid sequence can then be verified from such deposits.Moreover, the amino acid sequence of the protein encoded by a particularclone can also be directly determined by peptide sequencing or byexpressing the protein in a suitable host cell containing the depositedhuman cDNA, collecting the protein, and determining its sequence.

[0742] The present invention also relates to the genes corresponding toSEQ ID NO:X, SEQ ID NO:Y, or the deposited clone. The corresponding genecan be isolated in accordance with known methods using the sequenceinformation disclosed herein. Such methods include preparing probes orprimers from the disclosed sequence and identifying or amplifying thecorresponding gene from appropriate sources of genomic material.

[0743] Also provided in the present invention are allelic variants,orthologs, and/or species homologs. Procedures known in the art can beused to obtain full-length genes, allelic variants, splice variants,full-length coding portions, orthologs, and/or species homologs of genescorresponding to SEQ ID NO:X, SEQ ID NO:Y, or a deposited clone, usinginformation from the sequences disclosed herein or the clones depositedwith the ATCC. For example, allelic variants and/or species homologs maybe isolated and identified by making suitable probes or primers from thesequences provided herein and screening a suitable nucleic acid sourcefor allelic variants and/or the desired homologue.

[0744] The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0745] The polypeptides may be in the form of the secreted protein,including the mature form, or may be a part of a larger protein, such asa fusion protein (see below). It is often advantageous to include anadditional amino acid sequence which contains secretory or leadersequences, pro-sequences, sequences which aid in purification, such asmultiple histidine residues, or an additional sequence for stabilityduring recombinant production.

[0746] The polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, including the secretedpolypeptide, can be substantially purified using techniques describedherein or otherwise known in the art, such as, for example, by theone-step method described in Smith and Johnson, Gene 67:3140 (1988).Polypeptides of the invention also can be purified from natural,synthetic or recombinant sources using techniques described herein orotherwise known in the art, such as, for example, antibodies of theinvention raised against the secreted protein.

[0747] The present invention provides a polynucleotide comprising, oralternatively consisting of, the nucleic acid sequence of SEQ ID NO:X,and/or a cDNA contained in ATCC deposit Z. The present invention alsoprovides a polypeptide comprising, or alternatively, consisting of, thepolypeptide sequence of SEQ ID NO:Y and/or a polypeptide encoded by thecDNA contained in ATCC deposit Z. Polynucleotides encoding a polypeptidecomprising, or alternatively consisting of the polypeptide sequence ofSEQ ID NO:Y and/or a polypeptide sequence encoded by the cDNA containedin ATCC deposit Z are also encompassed by the invention.

[0748] Signal Sequences

[0749] The present invention also encompasses mature forms of thepolypeptide having the polypeptide sequence of SEQ ID NO:Y and/or thepolypeptide sequence encoded by the cDNA in a deposited clone.Polynucleotides encoding the mature forms (such as, for example, thepolynucleotide sequence in SEQ ID NO:X and/or the polynucleotidesequence contained in the cDNA of a deposited clone) are alsoencompassed by the invention. According to the signal hypothesis,proteins secreted by mammalian cells have a signal or secretary leadersequence which is cleaved from the mature protein once export of thegrowing protein chain across the rough endoplasmic reticulum has beeninitiated. Most mammalian cells and even insect cells cleave secretedproteins with the same specificity. However, in some cases, cleavage ofa secreted protein is not entirely uniform, which results in two or moremature species of the protein. Further, it has long been known thatcleavage specificity of a secreted protein is ultimately determined bythe primary structure of the complete protein, that is, it is inherentin the amino acid sequence of the polypeptide.

[0750] Methods for predicting whether a protein has a signal sequence,as well as the cleavage point for that sequence, are available. Forinstance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses theinformation from a short N-terminal charged region and a subsequentuncharged region of the complete (uncleaved) protein. The method of vonHeinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information fromthe residues surrounding the cleavage site, typically residues −13 to+2, where +1 indicates the amino terminus of the secreted protein. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80%. (vonHeinje, supra.) However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

[0751] In the present case, the deduced amino acid sequence of thesecreted polypeptide was analyzed by a computer program called SignalP(Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)), whichpredicts the cellular location of a protein based on the amino acidsequence. As part of this computational prediction of localization, themethods of McGeoch and von Heinje are incorporated. The analysis of theamino acid sequences of the secreted proteins described herein by thisprogram provided the results shown in Table 1.

[0752] As one of ordinary skill would appreciate, however, cleavagesites sometimes vary from organism to organism and cannot be predictedwith absolute certainty. Accordingly, the present invention providessecreted polypeptides having a sequence shown in SEQ ID NO:Y which havean N-terminus beginning within 5 residues (i.e., + or −5 residues) ofthe predicted cleavage point. Similarly, it is also recognized that insome cases, cleavage of the signal sequence from a secreted protein isnot entirely uniform, resulting in more than one secreted species. Thesepolypeptides, and the polynucleotides encoding such polypeptides, arecontemplated by the present invention.

[0753] Moreover, the signal sequence identified by the above analysismay not necessarily predict the naturally occurring signal sequence. Forexample, the naturally occurring signal sequence may be further upstreamfrom the predicted signal sequence. However, it is likely that thepredicted signal sequence will be capable of directing the secretedprotein to the ER. Nonetheless, the present invention provides themature protein produced by expression of the polynucleotide sequence ofSEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA ofa deposited clone, in a mammalian cell (e.g., COS cells, as describedbelow). These polypeptides, and the polynucleotides encoding suchpolypeptides, are contemplated by the present invention.

[0754] Polynucleotide and Polypeptide Variants

[0755] The present invention is directed to variants of thepolynucleotide sequence disclosed in SEQ ID NO:X, the complementarystrand thereto, and/or the cDNA sequence contained in a deposited clone.

[0756] The present invention also encompasses variants of thepolypeptide sequence disclosed in SEQ ID NO:Y and/or encoded by adeposited clone.

[0757] “Variant” refers to a polynucleotide or polypeptide differingfrom the polynucleotide or polypeptide of the present invention, butretaining essential properties thereof. Generally, variants are overallclosely similar, and, in many regions, identical to the polynucleotideor polypeptide of the present invention.

[0758] The present invention is also directed to nucleic acid moleculeswhich comprise, or alternatively consist of, a nucleotide sequence whichis at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forexample, the nucleotide coding sequence in SEQ ID NO:X or thecomplementary strand thereto, the nucleotide coding sequence containedin a deposited cDNA clone or the complementary strand thereto, anucleotide sequence encoding the polypeptide of SEQ ID NO:Y, anucleotide sequence encoding the polypeptide encoded by the cDNAcontained in a deposited clone, and/or polynucleotide fragments of anyof these nucleic acid molecules (e.g., those fragments describedherein). Polynucleotides which hybridize to these nucleic acid moleculesunder stringent hybridization conditions or lower stringency conditionsare also encompassed by the invention, as are polypeptides encoded bythese polynucleotides.

[0759] The present invention is also directed to polypeptides whichcomprise, or alternatively consist of, an amino acid sequence which isat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, forexample, the polypeptide sequence shown in SEQ ID NO:Y, the polypeptidesequence encoded by the cDNA contained in a deposited clone, and/orpolypeptide fragments of any of these polypeptides (e.g., thosefragments described herein).

[0760] By a nucleic acid having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence of thepresent invention, it is intended that the nucleotide sequence of thenucleic acid is identical to the reference sequence except that thenucleotide sequence may include up to five point mutations per each 100nucleotides of the reference nucleotide sequence encoding thepolypeptide. In other words, to obtain a nucleic acid having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence shown in Table 1, the ORF (open reading frame), orany fragment specified as described herein.

[0761] As a practical matter, whether any particular nucleic acidmolecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identical to a nucleotide sequence of the presence invention can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245(1990)). In a sequence alignment the query andsubject sequences are both DNA sequences. An RNA sequence can becompared by converting U's to T's. The result of said global sequencealignment is in percent identity. Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

[0762] If the subject sequence is shorter than the query sequencebecause of 5′ or 3′ deletions, not because of internal deletions, amanual correction must be made to the results. This is because theFASTDB program does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.Whether a nucleotide is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thiscorrected score is what is used for the purposes of the presentinvention. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score.

[0763] For example, a 90 base subject sequence is aligned to a 100 basequery sequence to determine percent identity. The deletions occur at the5′ end of the subject sequence and therefore, the FASTDB alignment doesnot show a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

[0764] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a query amino acid sequence of the presentinvention, it is intended that the amino acid sequence of the subjectpolypeptide is identical to the query sequence except that the subjectpolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a query amino acid sequence, up to 5% of the amino acidresidues in the subject sequence may be inserted, deleted, (indels) orsubstituted with another amino acid. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0765] As a practical matter, whether any particular polypeptide is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, an amino acid sequences shown in Table 1 (SEQ ID NO:Y) or tothe amino acid sequence encoded by cDNA contained in a deposited clonecan be determined conventionally using known computer programs. Apreferred method for determining the best overall match between a querysequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, can be determined usingthe FASTDB computer program based on the algorithm of Brutlag et al.(Comp. App. Biosci. 6:237-245(1990)). In a sequence alignment the queryand subject sequences are either both nucleotide sequences or both aminoacid sequences. The result of said global sequence alignment is inpercent identity. Preferred parameters used in a FASTDB amino acidalignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, JoiningPenalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter.

[0766] If the subject sequence is shorter than the query sequence due toN- or C-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

[0767] For example, a 90 amino acid residue subject sequence is alignedwith a 100 residue query sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the FASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

[0768] The variants may contain alterations in the coding regions,non-coding regions, or both. Especially preferred are polynucleotidevariants containing alterations which produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded polypeptide. Nucleotide variants produced by silentsubstitutions due to the degeneracy of the genetic code are preferred.Moreover, variants in which 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.Polynucleotide variants can be produced for a variety of reasons, e.g.,to optimize codon expression for a particular host (change codons in thehuman mRNA to those preferred by a bacterial host such as E. coli).

[0769] Naturally occurring variants are called “allelic variants,” andrefer to one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. (Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985).) These allelic variants can vary ateither the polynucleotide and/or polypeptide level and are included inthe present invention. Alternatively, non-naturally occurring variantsmay be produced by mutagenesis techniques or by direct synthesis.

[0770] Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the secreted protein without substantial loss ofbiological function. The authors of Ron et al., J. Biol. Chem. 268:2984-2988 (1993), reported variant KGF proteins having heparin bindingactivity even after deleting 3, 8, or 27 amino-terminal amino acidresidues. Similarly, Interferon gamma exhibited up to ten times higheractivity after deleting 8-10 amino acid residues from the carboxyterminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216(1988).)

[0771] Moreover, ample evidence demonstrates that variants often retaina biological activity similar to that of the naturally occurringprotein. For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111(1993)) conducted extensive mutational analysis of human cytokine IL-1a.They used random mutagenesis to generate over 3,500 individual IL-1amutants that averaged 2.5 amino acid changes per variant over the entirelength of the molecule. Multiple mutations were examined at everypossible amino acid position. The investigators found that “[m]ost ofthe molecule could be altered with little effect on either [binding orbiological activity].” (See, Abstract.) In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

[0772] Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

[0773] Thus, the invention further includes polypeptide variants whichshow substantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity. Forexample, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., Science 247:1306-1310(1990), wherein the authors indicate that there are two main strategiesfor studying the tolerance of an amino acid sequence to change.

[0774] The first strategy exploits the tolerance of amino acidsubstitutions by natural selection during the process of evolution. Bycomparing amino acid sequences in different species, conserved aminoacids can be identified. These conserved amino acids are likelyimportant for protein function. In contrast, the amino acid positionswhere substitutions have been tolerated by natural selection indicatesthat these positions are not critical for protein function. Thus,positions tolerating amino acid substitution could be modified whilestill maintaining biological activity of the protein.

[0775] The second strategy uses genetic engineering to introduce aminoacid changes at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

[0776] As the authors state, these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,most buried (within the tertiary structure of the protein) amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Moreover, tolerated conservativeamino acid substitutions involve replacement of the aliphatic orhydrophobic amino acids Ala, Val, Leu and Ile; replacement of thehydroxyl residues Ser and Thr; replacement of the acidic residues Aspand Glu; replacement of the amide residues Asn and Gln, replacement ofthe basic residues Lys, Arg, and His; replacement of the aromaticresidues Phe, Tyr, and Trp, and replacement of the small-sized aminoacids Ala, Ser, Thr, Met, and Gly.

[0777] Besides conservative amino acid substitution, variants of thepresent invention include (i) substitutions with one or more of thenon-conserved amino acid residues, where the substituted amino acidresidues may or may not be one encoded by the genetic code, or (ii)substitution with one or more of amino acid residues having asubstituent group, or (iii) fusion of the mature polypeptide withanother compound, such as a compound to increase the stability and/orsolubility of the polypeptide (for example, polyethylene glycol), or(iv) fusion of the polypeptide with additional amino acids, such as, forexample, an IgG Fc fusion region peptide, or leader or secretorysequence, or a sequence facilitating purification. Such variantpolypeptides are deemed to be within the scope of those skilled in theart from the teachings herein.

[0778] For example, polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

[0779] A further embodiment of the invention relates to a polypeptidewhich comprises the amino acid sequence of the present invention havingan amino acid sequence which contains at least one amino acidsubstitution, but not more than 50 amino acid substitutions, even morepreferably, not more than 40 amino acid substitutions, still morepreferably, not more than 30 amino acid substitutions, and still evenmore preferably, not more than 20 amino acid substitutions. Of course,in order of ever-increasing preference, it is highly preferable for apeptide or polypeptide to have an amino acid sequence which comprisesthe amino acid sequence of the present invention, which contains atleast one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidsubstitutions. In specific embodiments, the number of additions,substitutions, and/or deletions in the amino acid sequence of thepresent invention or fragments thereof (e.g., the mature form and/orother fragments described herein), is 1-5,5-10, 5-25, 5-50, 10-50 or50-150, conservative amino acid substitutions are preferable.

[0780] Polynucleotide and Polypeptide Fragments

[0781] The present invention is also directed to polynucleotidefragments of the polynucleotides of the invention.

[0782] In the present invention, a “polynucleotide fragment” refers to ashort polynucleotide having a nucleic acid sequence which: is a portionof that contained in a deposited clone, or encoding the polypeptideencoded by the cDNA in a deposited clone; is a portion of that shown inSEQ ID NO:X or the complementary strand thereto, or is a portion of apolynucleotide sequence encoding the polypeptide of SEQ ID NO:Y. Thenucleotide fragments of the invention are preferably at least about 15nt, and more preferably at least about 20 nt, still more preferably atleast about 30 nt, and even more preferably, at least about 40 nt, atleast about 50 nt, at least about 75 nt, or at least about 150 nt inlength. A fragment “at least 20 nt in length,” for example, is intendedto include 20 or more contiguous bases from the cDNA sequence containedin a deposited clone or the nucleotide sequence shown in SEQ ID NO:X. Inthis context “about” includes the particularly recited value, a valuelarger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini. These nucleotide fragments have uses thatinclude, but are not limited to, as diagnostic probes and primers asdiscussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600,2000 nucleotides) are preferred.

[0783] Moreover, representative examples of polynucleotide fragments ofthe invention, include, for example, fragments comprising, oralternatively consisting of, a sequence from about nucleotide number1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400,401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850,851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200,1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800,1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ IDNO:X, or the complementary strand thereto, or the cDNA contained in adeposited clone. In this context “about” includes the particularlyrecited ranges, and ranges larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini. Preferably,these fragments encode a polypeptide which has biological activity. Morepreferably, these polynucleotides can be used as probes or primers asdiscussed herein. Polynucleotides which hybridize to these nucleic acidmolecules under stringent hybridization conditions or lower stringencyconditions are also encompassed by the invention, as are polypeptidesencoded by these polynucleotides.

[0784] In the present invention, a “polypeptide fragment” refers to anamino acid sequence which is a portion of that contained in SEQ ID NO:Yor encoded by the cDNA contained in a deposited clone. Protein(polypeptide) fragments may be “free-standing,” or comprised within alarger polypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentscomprising, or alternatively consisting of, from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 tothe end of the coding region. Moreover, polypeptide fragments can beabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150amino acids in length. In this context “about” includes the particularlyrecited ranges or values, and ranges or values larger or smaller byseveral (5, 4, 3, 2, or 1) amino acids, at either extreme or at bothextremes. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0785] Preferred polypeptide fragments include the secreted protein aswell as the mature form. Further preferred polypeptide fragments includethe secreted protein or the mature form having a continuous series ofdeleted residues from the amino or the carboxy terminus, or both. Forexample, any number of amino acids, ranging from 1-60, can be deletedfrom the amino terminus of either the secreted polypeptide or the matureform. Similarly, any number of amino acids, ranging from 1-30, can bedeleted from the carboxy terminus of the secreted protein or matureform. Furthermore, any combination of the above amino and carboxyterminus deletions are preferred. Similarly, polynucleotides encodingthese polypeptide fragments are also preferred.

[0786] Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:Y falling withinconserved domains are specifically contemplated by the presentinvention. Moreover, polynucleotides encoding these domains are alsocontemplated.

[0787] Other preferred polypeptide fragments are biologically activefragments. Biologically active fragments are those exhibiting activitysimilar, but not necessarily identical, to an activity of thepolypeptide of the present invention. The biological activity of thefragments may include an improved desired activity, or a decreasedundesirable activity. Polynucleotides encoding these polypeptidefragments are also encompassed by the invention.

[0788] Preferably, the polynucleotide fragments of the invention encodea polypeptide which demonstrates a functional activity. By a polypeptidedemonstrating a “functional activity” is meant, a polypeptide capable ofdisplaying one or more known functional activities associated with afull-length (complete) polypeptide of invention protein. Such functionalactivities include, but are not limited to, biological activity,antigenicity [ability to bind (or compete with a polypeptide of theinvention for binding) to an antibody to the polypeptide of theinvention], immunogenicity (ability to generate antibody which binds toa polypeptide of the invention), ability to form multimers withpolypeptides of the invention, and ability to bind to a receptor orligand for a polypeptide of the invention.

[0789] The functional activity of polypeptides of the invention, andfragments, variants derivatives, and analogs thereof, can be assayed byvarious methods.

[0790] For example, in one embodiment where one is assaying for theability to bind or compete with full-length polypeptide of the inventionfor binding to an antibody of the polypeptide of the invention, variousimmunoassays known in the art can be used, including but not limited to,competitive and non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

[0791] In another embodiment, where a ligand for a polypeptide of theinvention identified, or the ability of a polypeptide fragment, variantor derivative of the invention to multimerize is being evaluated,binding can be assayed, e.g., by means well-known in the art, such as,for example, reducing and non-reducing gel chromatography, proteinaffinity chromatography, and affinity blotting. See generally, Phizicky,E., et al., 1995, Microbiol. Rev. 59:94-123. In another embodiment,physiological correlates of binding of a polypeptide of the invention toits substrates (signal transduction) can be assayed.

[0792] In addition, assays described herein (see Examples) and otherwiseknown in the art may routinely be applied to measure the ability ofpolypeptides of the invention and fragments, variants derivatives andanalogs thereof to elicit related biological activity related to that ofthe polypeptide of the invention (either in vitro or in vivo). Othermethods will be known to the skilled artisan and are within the scope ofthe invention.

[0793] Epitopes and Antibodies

[0794] The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:Y, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in ATCC depositNo. Z or encoded by a polynucleotide that hybridizes to the complementof the sequence of SEQ ID NO:X or contained in ATCC deposit No. Z understringent hybridization conditions or lower stringency hybridizationconditions as defined supra. The present invention further encompassespolynucleotide sequences encoding an epitope of a polypeptide sequenceof the invention (such as, for example, the sequence disclosed in SEQ IDNO:X), polynucleotide sequences of the complementary strand of apolynucleotide sequence encoding an epitope of the invention, andpolynucleotide sequences which hybridize to the complementary strandunder stringent hybridization conditions or lower stringencyhybridization conditions defined supra.

[0795] The term “epitopes,” as used herein, refers to portions of apolypeptide having antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably in a human. In a preferredembodiment, the present invention encompasses a polypeptide comprisingan epitope, as well as the polynucleotide encoding this polypeptide. An“immunogenic epitope,” as used herein, is defined as a portion of aprotein that elicits an antibody response in an animal, as determined byany method known in the art, for example, by the methods for generatingantibodies described infra. (See, for example, Geysen et al., Proc.Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,”as used herein, is defined as a portion of a protein to which anantibody can immunospecifically bind its antigen as determined by anymethod well known in the art, for example, by the immunoassays describedherein. Immunospecific binding excludes non-specific binding but doesnot necessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

[0796] Fragments which function as epitopes may be produced by anyconventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

[0797] In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof. Antigenic epitopes are useful, for example, to raiseantibodies, including monoclonal antibodies, that specifically bind theepitope. Preferred antigenic epitopes include the antigenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes can be used asthe target molecules in immunoassays. (See, for instance, Wilson et al.,Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

[0798] Similarly, immunogenic epitopes can be used, for example, toinduce antibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

[0799] Epitope-bearing polypeptides of the present invention may be usedto induce antibodies according to methods well known in the artincluding, but not limited to, in vivo immunization, in vitroimmunization, and phage display methods. See, e.g., Sutcliffe et al.,supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol.,66:2347-2354 (1985). If in vivo immunization is used, animals may beimmunized with free peptide; however, anti-peptide antibody titer may beboosted by coupling the peptide to a macromolecular carrier, such askeyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance,peptides containing cysteine residues may be coupled to a carrier usinga linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),while other peptides may be coupled to carriers using a more generallinking agent such as glutaraldehyde. Animals such as rabbits, rats andmice are immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 μg of peptide or carrier protein and Freund'sadjuvant or any other adjuvant known for stimulating an immune response.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

[0800] As one of skill in the art will appreciate, and as discussedabove, the polypeptides of the present invention comprising animmunogenic or antigenic epitope can be fused to other polypeptidesequences. For example, the polypeptides of the present invention may befused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM),or portions thereof (CH1, CH2, CH3, or any combination thereof andportions thereof) resulting in chimeric polypeptides. Such fusionproteins may facilitate purification and may increase half-life in vivo.This has been shown for chimeric proteins consisting of the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins. See,e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanceddelivery of an antigen across the epithelial barrier to the immunesystem has been demonstrated for antigens (e.g., insulin) conjugated toan FcRn binding partner such as IgG or Fc fragments (see, e.g., PCTPublications WO 96/22024 and WO 99/04813). IgG Fusion proteins that havea disulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed) by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded ontoNi2+nitriloacetic acid-agarose column and histidine-tagged proteins canbe selectively eluted with imidazole-containing buffers.

[0801] Additional fusion proteins of the invention may be generatedthrough the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling may be employed to modulate the activities ofpolypeptides of the invention, such methods can be used to generatepolypeptides with altered activity, as well as agonists and antagonistsof the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793;5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr.Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol.16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999);and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of thesepatents and publications are hereby incorporated by reference in itsentirety). In one embodiment, alteration of polynucleotidescorresponding to SEQ ID NO:X and the polypeptides encoded by thesepolynucleotides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments by homologous or site-specificrecombination to generate variation in the polynucleotide sequence. Inanother embodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

[0802] Antibodies

[0803] Further polypeptides of the invention relate to antibodies andT-cell antigen receptors (TCR) which immunospecifically bind apolypeptide, polypeptide fragment, or variant of SEQ ID NO:Y, and/or anepitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0804] Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)₂, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

[0805] The antibodies of the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt,et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

[0806] Antibodies of the present invention may be described or specifiedin terms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

[0807] Antibodies of the present invention may also be described orspecified in terms of their cross-reactivity. Antibodies that do notbind any other analog, ortholog, or homolog of a polypeptide of thepresent invention are included. Antibodies that bind polypeptides withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. In specific embodiments, antibodies of thepresent invention cross-react with murine, rat and/or rabbit homologs ofhuman proteins and the corresponding epitopes thereof. Antibodies thatdo not bind polypeptides with less than 95%, less than 90%, less than85%, less than 80%, less than 75%, less than 70%, less than 65%, lessthan 60%, less than 55%, and less than 50% identity (as calculated usingmethods known in the art and described herein) to a polypeptide of thepresent invention are also included in the present invention. In aspecific embodiment, the above-described cross-reactivity is withrespect to any single specific antigenic or immunogenic polypeptide, orcombination(s) of 2, 3, 4, 5, or more of the specific antigenic and/orimmunogenic polypeptides disclosed herein. Further include/d in thepresent invention are antibodies which bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity to a polypeptide of theinvention. Preferred binding affinities include those with adissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³M, 5×10⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁻⁷ M,5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵M, or 10⁻¹⁵ M.

[0808] The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

[0809] Antibodies of the present invention may act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Preferably, antibodies of the presentinvention bind an antigenic epitope disclosed herein, or a portionthereof. The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand activity or receptor activity by atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50% of the activity in absence ofthe antibody.

[0810] The invention also features receptor-specific antibodies whichboth prevent ligand binding and receptor activation as well asantibodies that recognize the receptor-ligand complex, and, preferably,do not specifically recognize the unbound receptor or the unboundligand. Likewise, included in the invention are neutralizing antibodieswhich bind the ligand and prevent binding of the ligand to the receptor,as well as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

[0811] Antibodies of the present invention may be used, for example, butnot limited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

[0812] As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

[0813] The antibodies of the invention include derivatives that aremodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom generating an anti-idiotypic response. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

[0814] The antibodies of the present invention may be generated by anysuitable method known in the art. Polyclonal antibodies to anantigen-of-interest can be produced by various procedures well known inthe art. For example, a polypeptide of the invention can be administeredto various host animals including, but not limited to, rabbits, mice,rats, etc. to induce the production of sera containing polyclonalantibodies specific for the antigen. Various adjuvants may be used toincrease the immunological response, depending on the host species, andinclude but are not limited to, Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

[0815] Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

[0816] Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples (e.g., Example 16). In anon-limiting example, mice can be immunized with a polypeptide of theinvention or a cell expressing such peptide. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theATCC. Hybridomas are selected and cloned by limited dilution. Thehybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding a polypeptide of theinvention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

[0817] Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

[0818] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, Fab and F(ab′)₂ fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). F(ab′)₂ fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

[0819] For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

[0820] As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJR134:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

[0821] Examples of techniques which can be used to produce single-chainFvs and antibodies include those described in U.S. Pat. Nos. 4,946,778and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991);Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science240:1038-1040 (1988). For some uses, including in vivo use of antibodiesin humans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. A chimeric antibody is amolecule in which different portions of the antibody are derived fromdifferent animal species, such as antibodies having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region. Methods for producing chimeric antibodies are known inthe art. See e.g., Morrison, Science 229:1202 (1985); Oi et al.,BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, whichare incorporated herein by reference in their entirety. Humanizedantibodies are antibody molecules from non-human species antibody thatbinds the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and a framework regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

[0822] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which isincorporated herein by reference in its entirety.

[0823] Human antibodies can also be produced using transgenic mice whichare incapable of expressing functional endogenous immunoglobulins, butwhich can express human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and Genpharrn (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

[0824] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

[0825] Further, antibodies to the polypeptides of the invention can, inturn, be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

[0826] Polynucleotides Encoding Antibodies

[0827] The invention further provides polynucleotides comprising anucleotide sequence encoding an antibody of the invention and fragmentsthereof. The invention also encompasses polynucleotides that hybridizeunder stringent or lower stringency hybridization conditions, e.g., asdefined supra, to polynucleotides that encode an antibody, preferably,that specifically binds to a polypeptide of the invention, preferably,an antibody that binds to a polypeptide having the amino acid sequenceof SEQ ID NO:Y.

[0828] The polynucleotides may be obtained, and the nucleotide sequenceof the polynucleotides determined, by any method known in the art. Forexample, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,BioTechniques 17:242 (1994)), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

[0829] Alternatively, a polynucleotide encoding an antibody may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the immunoglobulin may be chemically synthesized orobtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody of the invention) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method well known in the art.

[0830] Once the nucleotide sequence and corresponding amino acidsequence of the antibody is determined, the nucleotide sequence of theantibody may be manipulated using methods well known in the art for themanipulation of nucleotide sequences, e.g., recombinant DNA techniques,site directed mutagenesis, PCR, etc. (see, for example, the techniquesdescribed in Sambrook et al., 1990, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties), to generate antibodies having a different aminoacid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

[0831] In a specific embodiment, the amino acid sequence of the heavyand/or light chain variable domains may be inspected to identify thesequences of the complementarity determining regions (CDRs) by methodsthat are well know in the art, e.g., by comparison to known amino acidsequences of other heavy and light chain variable regions to determinethe regions of sequence hypervariability. Using routine recombinant DNAtechniques, one or more of the CDRs may be inserted within frameworkregions, e.g., into human framework regions to humanize a non-humanantibody, as described supra. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998)for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

[0832] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

[0833] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42(1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);and Ward et al., Nature 334:544-54 (1989)) can be adapted to producesingle chain antibodies. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242:1038-1041 (1988)).

[0834] Methods of Producing Antibodies

[0835] The antibodies of the invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

[0836] Recombinant expression of an antibody of the invention, orfragment, derivative or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

[0837] The expression vector is transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody of the invention. Thus,the invention includes host cells containing a polynucleotide encodingan antibody of the invention, or a heavy or light chain thereof, or asingle chain antibody of the invention, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

[0838] A variety of host-expression vector systems may be utilized toexpress the antibody molecules of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody molecule of the invention in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing antibodycoding sequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

[0839] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

[0840] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The antibody coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

[0841] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the antibody coding sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the antibody molecule in infectedhosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359(1984)). Specific initiation signals may also be required for efficienttranslation of inserted antibody coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see Bittner et al.,Methods in Enzymol. 153:51-544 (1987)).

[0842] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

[0843] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the antibody molecule may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

[0844] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

[0845] The expression levels of an antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol.3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

[0846] The host cell may be co-transfected with two expression vectorsof the invention, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector may be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52(1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The codingsequences for the heavy and light chains may comprise cDNA or genomicDNA.

[0847] Once an antibody molecule of the invention has been produced byan animal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

[0848] The present invention encompasses antibodies recombinantly fusedor chemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

[0849] The present invention further includes compositions comprisingthe polypeptides of the present invention fused or conjugated toantibody domains other than the variable regions. For example, thepolypeptides of the present invention may be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention may comprise the constant region,hinge region, CH1 domain, CH2 domain, and CH3 domain or any combinationof whole domains or portions thereof. The polypeptides may also be fusedor conjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

[0850] As discussed, supra, the polypeptides corresponding to apolypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides corresponding to SEQID NO:Y may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson etal., J. Biol. Chem. 270:9459-9471 (1995).

[0851] Moreover, the antibodies or fragments thereof of the presentinvention can be fused to marker sequences, such as a peptide tofacilitate purification. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., Cell37:767 (1984)) and the “flag” tag.

[0852] The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, 111In or 99Tc.

[0853] Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0854] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, B-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0855] Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

[0856] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

[0857] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980, which is incorporated herein by reference in itsentirety.

[0858] An antibody, with or without a therapeutic moiety conjugated toit, administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

[0859] Immunophenotyping

[0860] The antibodies of the invention may be utilized forimmunophenotyping of cell lines and biological samples. The translationproduct of the gene of the present invention may be useful as a cellspecific marker, or more specifically as a cellular marker that isdifferentially expressed at various stages of differentiation and/ormaturation of particular cell types. Monoclonal antibodies directedagainst a specific epitope, or combination of epitopes, will allow forthe screening of cellular populations expressing the marker. Varioustechniques can be utilized using monoclonal antibodies to screen forcellular populations expressing the marker(s), and include magneticseparation using antibody-coated magnetic beads, “panning” with antibodyattached to a solid matrix (i.e., plate), and flow cytometry (See, e.g.,U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

[0861] These techniques allow for the screening of particularpopulations of cells, such as might be found with hematologicalmalignancies (i.e. minimal residual disease (MRD) in acute leukemicpatients) and “non-self” cells in transplantations to preventGraft-versus-Host Disease (GVHD). Alternatively, these techniques allowfor the screening of hematopoietic stem and progenitor cells capable ofundergoing proliferation and/or differentiation, as might be found inhuman umbilical cord blood.

[0862] Assays for Antibody Binding

[0863] The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

[0864] Immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIPA buffer (1% NP-40 orTriton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trasylol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1-4 hours) at 4° C., addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 4° C., washing the beads inlysis buffer and resuspending the beads in SDS/sample buffer. Theability of the antibody of interest to immunoprecipitate a particularantigen can be assessed by, e.g., western blot analysis. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0865] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blocking themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., 32P or 125I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0866] ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

[0867] The binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., 3H or 125I) with the antibody of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofinterest for a particular antigen and the binding off-rates can bedetermined from the data by scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, the antigen is incubated with antibody of interest conjugated to alabeled compound (e.g., 3H or 125I) in the presence of increasingamounts of an unlabeled second antibody.

[0868] Therapeutic Uses

[0869] The present invention is further directed to antibody-basedtherapies which involve administering antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, patient fortreating one or more of the disclosed diseases, disorders, orconditions. Therapeutic compounds of the invention include, but are notlimited to, antibodies of the invention (including fragments, analogsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

[0870] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0871] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

[0872] The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

[0873] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵M, 5×10⁻⁶ M,10⁻⁶M, 5×10⁻⁷M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

[0874] Gene Therapy

[0875] In a specific embodiment, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of a polypeptide of the invention,by way of gene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

[0876] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0877] For general reviews of the methods of gene therapy, see Goldspielet al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596(1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0878] In a preferred aspect, the compound comprises nucleic acidsequences encoding an antibody, said nucleic acid sequences being partof expression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

[0879] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid-carrying vectors, or indirect, in which case, cellsare first transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

[0880] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

[0881] In a specific embodiment, viral vectors that contains nucleicacid sequences encoding an antibody of the invention are used. Forexample, a retroviral vector can be used (see Miller et al., Meth.Enzymol. 217:581-599 (1993)). These retroviral vectors contain thecomponents necessary for the correct packaging of the viral genome andintegration into the host cell DNA. The nucleic acid sequences encodingthe antibody to be used in gene therapy are cloned into one or morevectors, which facilitates delivery of the gene into a patient. Moredetail about retroviral vectors can be found in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993).

[0882] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

[0883] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300(1993); U.S. Pat. No. 5,436,146).

[0884] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

[0885] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0886] The resulting recombinant cells can be delivered to a patient byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

[0887] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0888] In a preferred embodiment, the cell used for gene therapy isautologous to the patient.

[0889] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding an antibody are introduced intothe cells such that they are expressible by the cells or their progeny,and the recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

[0890] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription. Demonstration of Therapeutic or ProphylacticActivity

[0891] The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

[0892] Therapeutic/Prophylactic Administration and Composition

[0893] The invention provides methods of treatment, inhibition andprophylaxis by administration to a subject of an effective amount of acompound or pharmaceutical composition of the invention, preferably anantibody of the invention. In a preferred aspect, the compound issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects). The subject ispreferably an animal, including but not limited to animals such as cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human.

[0894] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid or an immunoglobulin aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

[0895] Various delivery systems are known and can be used to administera compound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

[0896] In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

[0897] In another embodiment, the compound or composition can bedelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

[0898] In yet another embodiment, the compound or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989)). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

[0899] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0900] In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

[0901] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of acompound, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0902] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0903] The compounds of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0904] The amount of the compound of the invention which will beeffective in the treatment, inhibition and prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

[0905] For antibodies, the dosage administered to a patient is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

[0906] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. Diagnosis and Imaging

[0907] Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of aberrant expression.

[0908] The invention provides a diagnostic assay for diagnosing adisorder, comprising (a) assaying the expression of the polypeptide ofinterest in cells or body fluid of an individual using one or moreantibodies specific to the polypeptide interest and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a particulardisorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0909] Antibodies of the invention can be used to assay protein levelsin a biological sample using classical immunohistological methods knownto those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

[0910] One aspect of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of a polypeptideof interest in an animal, preferably a mammal and most preferably ahuman. In one embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled molecule which specificallybinds to the polypeptide of interest; b) waiting for a time intervalfollowing the administering for permitting the labeled molecule topreferentially concentrate at sites in the subject where the polypeptideis expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled molecule in the subject, such that detection of labeled moleculeabove the background level indicates that the subject has a particulardisease or disorder associated with aberrant expression of thepolypeptide of interest. Background level can be determined by variousmethods including, comparing the amount of labeled molecule detected toa standard value previously determined for a particular system.

[0911] It will be understood in the art that the size of the subject andthe imaging system used will determine the quantity of imaging moietyneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of 99 mTc. The labeledantibody or antibody fragment will then preferentially accumulate at thelocation of cells which contain the specific protein. In vivo tumorimaging is described in S. W. Burchiel et al., “Immunopharmacokineticsof Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

[0912] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0913] In an embodiment, monitoring of the disease or disorder iscarried out by repeating the method for diagnosing the disease ordisease, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

[0914] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

[0915] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI). Kits

[0916] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

[0917] In another specific embodiment of the present invention, the kitis a diagnostic kit for use in screening serum containing antibodiesspecific against proliferative and/or cancerous polynucleotides andpolypeptides. Such a kit may include a control antibody that does notreact with the polypeptide of interest. Such a kit may include asubstantially isolated polypeptide antigen comprising an epitope whichis specifically immunoreactive with at least one anti-polypeptideantigen antibody. Further, such a kit includes means for detecting thebinding of said antibody to the antigen (e.g., the antibody may beconjugated to a fluorescent compound such as fluorescein or rhodaminewhich can be detected by flow cytometry). In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedpolypeptide antigen. The polypeptide antigen of the kit may also beattached to a solid support.

[0918] In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which said polypeptideantigen is attached. Such a kit may also include a non-attachedreporter-labeled anti-human antibody. In this embodiment, binding of theantibody to the polypeptide antigen can be detected by binding of thesaid reporter-labeled antibody.

[0919] In an additional embodiment, the invention includes a diagnostickit for use in screening serum containing antigens of the polypeptide ofthe invention. The diagnostic kit includes a substantially isolatedantibody specifically immunoreactive with polypeptide or polynucleotideantigens, and means for detecting the binding of the polynucleotide orpolypeptide antigen to the antibody. In one embodiment, the antibody isattached to a solid support. In a specific embodiment, the antibody maybe a monoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

[0920] In one diagnostic configuration, test serum is reacted with asolid phase reagent having a surface-bound antigen obtained by themethods of the present invention. After binding with specific antigenantibody to the reagent and removing unbound serum components bywashing, the reagent is reacted with reporter-labeled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti-antigen antibody on the solid support. The reagent is againwashed to remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

[0921] The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

[0922] Thus, the invention provides an assay system or kit for carryingout this diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

[0923] Fusion Proteins

[0924] Any polypeptide of the present invention can be used to generatefusion proteins. For example, the polypeptide of the present invention,when fused to a second protein, can be used as an antigenic tag.Antibodies raised against the polypeptide of the present invention canbe used to indirectly detect the second protein by binding to thepolypeptide. Moreover, because secreted proteins target cellularlocations based on trafficking signals, the polypeptides of the presentinvention can be used as targeting molecules once fused to otherproteins.

[0925] Examples of domains that can be fused to polypeptides of thepresent invention include not only heterologous signal sequences, butalso other heterologous functional regions. The fusion does notnecessarily need to be direct, but may occur through linker sequences.

[0926] Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Also, peptide moieties may be added tothe polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routinetechniques in the art.

[0927] Moreover, polypeptides of the present invention, includingfragments, and specifically epitopes, can be combined with parts of theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portionsthereof (CH1, CH2, CH3, and any combination thereof, including bothentire domains and portions thereof), resulting in chimericpolypeptides. These fusion proteins facilitate purification and show anincreased half-life in vivo. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP A 394,827; Trauneckeret al., Nature 331:84-86 (1988).) Fusion proteins havingdisulfide-linked dimeric structures (due to the IgG) can also be moreefficient in binding and neutralizing other molecules, than themonomeric secreted protein or protein fragment alone. (Fountoulakis etal., J. Biochem. 270:3958-3964 (1995).)

[0928] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

[0929] Moreover, the polypeptides of the present invention can be fusedto marker sequences, such as a peptide which facilitates purification ofthe fused polypeptide. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Another peptide tag useful for purification, the “HA”tag, corresponds to an epitope derived from the influenza hemagglutininprotein. (Wilson et al., Cell 37:767 (1984).)

[0930] Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

[0931] Vectors, Host Cells, and Protein Production

[0932] The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

[0933] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0934] The polynucleotide insert should be operatively linked to anappropriate promoter, such as the phage lambda PL promoter, the E. colilac, trp, phoA and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, to name a few. Other suitable promoterswill be known to the skilled artisan. The expression constructs willfurther contain sites for transcription initiation, termination, and, inthe transcribed region, a ribosome binding site for translation. Thecoding portion of the transcripts expressed by the constructs willpreferably include a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

[0935] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase, G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

[0936] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223⁻³, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Other suitable vectors will be readily apparent to the skilled artisan.

[0937] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking arecombinant vector.

[0938] A polypeptide of this invention can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

[0939] Polypeptides of the present invention, and preferably thesecreted form, can also be recovered from: products purified fromnatural sources, including bodily fluids, tissues and cells, whetherdirectly isolated or cultured; products of chemical syntheticprocedures; and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial,yeast, higher plant, insect, and mammalian cells. Depending upon thehost employed in a recombinant production procedure, the polypeptides ofthe present invention may be glycosylated or may be non-glycosylated. Inaddition, polypeptides of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins, thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

[0940] In addition to encompassing host cells containing the vectorconstructs discussed herein, the invention also encompasses primary,secondary, and immortalized host cells of vertebrate origin,particularly mammalian origin, that have been engineered to delete orreplace endogenous genetic material (e.g., coding sequence), and/or toinclude genetic material (e.g., heterologous polynucleotide sequences)that is operably associated with the polynucleotides of the invention,and which activates, alters, and/or amplifies endogenouspolynucleotides. For example, techniques known in the art may be used tooperably associate heterologous control regions (e.g., promoter and/orenhancer) and endogenous polynucleotide sequences via homologousrecombination, resulting in the formation of a new transcription unit(see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No.5,733,761, issued Mar. 31, 1998; International Publication No. WO96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989),the disclosures of each of which are incorporated by reference in theirentireties).

[0941] In addition, polypeptides of the invention can be chemicallysynthesized using techniques known in the art (e.g., see Creighton,1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co.,N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example,a polypeptide corresponding to a fragment of a polypeptide sequence ofthe invention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thepolypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

[0942] The invention encompasses polypeptides which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited, to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

[0943] Additional post-translational modifications encompassed by theinvention include, for example, e.g., N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

[0944] Also provided by the invention are chemically modifiedderivatives of the polypeptides of the invention which may provideadditional advantages such as increased solubility, stability andcirculating time of the polypeptide, or decreased immunogenicity (seeU.S. Pat. No. 4,179,337). The chemical moieties for derivitization maybe selected from water soluble polymers such as polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, polyvinyl alcohol and the like. The polypeptides may bemodified at random positions within the molecule, or at predeterminedpositions within the molecule and may include one, two, three or moreattached chemical moieties.

[0945] The polymer may be of any molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 1 kDa and about 100 kDa (the term “about” indicatingthat in preparations of polyethylene glycol, some molecules will weighmore, some less, than the stated molecular weight) for ease in handlingand manufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

[0946] The polyethylene glycol molecules (or other chemical moieties)should be attached to the protein with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384, herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

[0947] One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

[0948] The polypeptides of the invention may be in monomers or multimers(i.e., dimers, trimers, tetramers and higher multimers). Accordingly,the present invention relates to monomers and multimers of thepolypeptides of the invention, their preparation, and compositions(preferably, Therapeutics) containing them. In specific embodiments, thepolypeptides of the invention are monomers, dimers, trimers ortetramers. In additional embodiments, the multimers of the invention areat least dimers, at least trimers, or at least tetramers.

[0949] Multimers encompassed by the invention may be homomers orheteromers. As used herein, the term homomer, refers to a multimercontaining only polypeptides corresponding to the amino acid sequence ofSEQ ID NO:Y or encoded by the cDNA contained in a deposited clone(including fragments, variants, splice variants, and fusion proteins,corresponding to these polypeptides as described herein). These homomersmay contain polypeptides having identical or different amino acidsequences. In a specific embodiment, a homomer of the invention is amultimer containing only polypeptides having an identical amino acidsequence. In another specific embodiment, a homomer of the invention isa multimer containing polypeptides having different amino acidsequences. In specific embodiments, the multimer of the invention is ahomodimer (e.g., containing polypeptides having identical or differentamino acid sequences) or a homotrimer (e.g., containing polypeptideshaving identical and/or different amino acid sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

[0950] As used herein, the term heteromer refers to a multimercontaining one or more heterologous polypeptides (i.e., polypeptides ofdifferent proteins) in addition to the polypeptides of the invention. Ina specific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

[0951] Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

[0952] In one example, covalent associations are between theheterologous sequence contained in a fusion protein of the invention(see, e.g., U.S. Pat. No. 5,478,925). In a specific example, thecovalent associations are between the heterologous sequence contained inan Fc fusion protein of the invention (as described herein). In anotherspecific example, covalent associations of fusion proteins of theinvention are between heterologous polypeptide sequence from anotherprotein that is capable of forming covalently associated multimers, suchas for example, oseteoprotegerin (see, e.g., International PublicationNO: WO 98/49305, the contents of which are herein incorporated byreference in its entirety). In another embodiment, two or morepolypeptides of the invention are joined through peptide linkers.Examples include those peptide linkers described in U.S. Pat. No.5,073,627 (hereby incorporated by reference). Proteins comprisingmultiple polypeptides of the invention separated by peptide linkers maybe produced using conventional recombinant DNA technology.

[0953] Another method for preparing multimer polypeptides of theinvention involves use of polypeptides of the invention fused to aleucine zipper or isoleucine zipper polypeptide sequence. Leucine zipperand isoleucine zipper domains are polypeptides that promotemultimerization of the proteins in which they are found. Leucine zipperswere originally identified in several DNA-binding proteins (Landschulzet al., Science 240:1759, (1988)), and have since been found in avariety of different proteins. Among the known leucine zippers arenaturally occurring peptides and derivatives thereof that dimerize ortrimerize. Examples of leucine zipper domains suitable for producingsoluble multimeric proteins of the invention are those described in PCTapplication WO 94/10308, hereby incorporated by reference. Recombinantfusion proteins comprising a polypeptide of the invention fused to apolypeptide sequence that dimerizes or trimerizes in solution areexpressed in suitable host cells, and the resulting soluble multimericfusion protein is recovered from the culture supernatant usingtechniques known in the art.

[0954] Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

[0955] In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in Flag® fusion proteins of the invention and anti-Flag®antibody.

[0956] The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

[0957] Alternatively, multimers of the invention may be generated usinggenetic engineering techniques known in the art. In one embodiment,polypeptides contained in multimers of the invention are producedrecombinantly using fusion protein technology described herein orotherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety). In a specificembodiment, polynucleotides coding for a homodimer of the invention aregenerated by ligating a polynucleotide sequence encoding a polypeptideof the invention to a sequence encoding a linker polypeptide and thenfurther to a synthetic polynucleotide encoding the translated product ofthe polypeptide in the reverse orientation from the original C-terminusto the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.No. 5,478,925, which is herein incorporated by reference in itsentirety). In another embodiment, recombinant techniques describedherein or otherwise known in the art are applied to generate recombinantpolypeptides of the invention which contain a transmembrane domain (orhydrophobic or signal peptide) and which can be incorporated by membranereconstitution techniques into liposomes (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).

[0958] Uses of the Polynucleotides

[0959] Each of the polynucleotides identified herein can be used innumerous ways as reagents. The following description should beconsidered exemplary and utilizes known techniques.

[0960] The polynucleotides of the present invention are useful forchromosome identification. There exists an ongoing need to identify newchromosome markers, since few chromosome marking reagents, based onactual sequence data (repeat polymorphisms), are presently available.Each polynucleotide of the present invention can be used as a chromosomemarker.

[0961] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:X.Primers can be selected using computer analysis so that primers do notspan more than one predicted exon in the genomic DNA. These primers arethen used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humangene corresponding to the SEQ ID NO:X will yield an amplified fragment.

[0962] Similarly, somatic hybrids provide a rapid method of PCR mappingthe polynucleotides to particular chromosomes. Three or more clones canbe assigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeledflow-sorted chromosomes, and preselection by hybridization to constructchromosome specific-cDNA libraries.

[0963] Precise chromosomal location of the polynucleotides can also beachieved using fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,0004,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

[0964] For chromosome mapping, the polynucleotides can be usedindividually (to mark a single chromosome or a single site on thatchromosome) or in panels (for marking multiple sites and/or multiplechromosomes). Preferred polynucleotides correspond to the noncodingregions of the cDNAs because the coding sequences are more likelyconserved within gene families, thus increasing the chance of crosshybridization during chromosomal mapping.

[0965] Once a polynucleotide has been mapped to a precise chromosomallocation, the physical position of the polynucleotide can be used inlinkage analysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. (Diseasemapping data are found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library).) Assuming 1 megabase mapping resolution and onegene per 20 kb, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50-500 potential causativegenes.

[0966] Thus, once coinheritance is established, differences in thepolynucleotide and the corresponding gene between affected andunaffected individuals can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected individuals, but not in normalindividuals, indicates that the mutation may cause the disease. However,complete sequencing of the polypeptide and the corresponding gene fromseveral normal individuals is required to distinguish the mutation froma polymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

[0967] Furthermore, increased or decreased expression of the gene inaffected individuals as compared to unaffected individuals can beassessed using polynucleotides of the present invention. Any of thesealterations (altered expression, chromosomal rearrangement, or mutation)can be used as a diagnostic or prognostic marker.

[0968] Thus, the invention also provides a diagnostic method usefulduring diagnosis of a disorder, involving measuring the expression levelof polynucleotides of the present invention in cells or body fluid froman individual and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

[0969] In still another embodiment, the invention includes a kit foranalyzing samples for the presence of proliferative and/or cancerouspolynucleotides derived from a test subject. In a general embodiment,the kit includes at least one polynucleotide probe containing anucleotide sequence that will specifically hybridize with apolynucleotide of the present invention and a suitable container. In aspecific embodiment, the kit includes two polynucleotide probes definingan internal region of the polynucleotide of the present invention, whereeach probe has one strand containing a 31′mer-end internal to theregion. In a further embodiment, the probes may be useful as primers forpolymerase chain reaction amplification.

[0970] Where a diagnosis of a disorder, has already been made accordingto conventional methods, the present invention is useful as a prognosticindicator, whereby patients exhibiting enhanced or depressedpolynucleotide of the present invention expression will experience aworse clinical outcome relative to patients expressing the gene at alevel nearer the standard level.

[0971] By “measuring the expression level of polynucleotide of thepresent invention” is intended qualitatively or quantitatively measuringor estimating the level of the polypeptide of the present invention orthe level of the mRNA encoding the polypeptide in a first biologicalsample either directly (e.g., by determining or estimating absoluteprotein level or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the disorder or beingdetermined by averaging levels from a population of individuals nothaving a disorder. As will be appreciated in the art, once a standardpolypeptide level or mRNA level is known, it can be used repeatedly as astandard for comparison.

[0972] By “biological sample” is intended any biological sample obtainedfrom an individual, body fluid, cell line, tissue culture, or othersource which contains the polypeptide of the present invention or mRNA.As indicated, biological samples include body fluids (such as semen,lymph, sera, plasma, urine, synovial fluid and spinal fluid) whichcontain the polypeptide of the present invention, and other tissuesources found to express the polypeptide of the present invention.Methods for obtaining tissue biopsies and body fluids from mammals arewell known in the art. Where the biological sample is to include mRNA, atissue biopsy is the preferred source.

[0973] The method(s) provided above may preferably be applied in adiagnostic method and/or kits in which polynucleotides and/orpolypeptides are attached to a solid support. In one exemplary method,the support may be a “gene chip” or a “biological chip” as described inU.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a genechip with polynucleotides of the present invention attached may be usedto identify polymorphisms between the polynucleotide sequences, withpolynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) wouldbe beneficial in identifying disease loci for many disorders, includingcancerous diseases and conditions. Such a method is described in U.S.Pat. Nos. 5,858,659 and 5,856,104. The US Patents referenced supra arehereby incorporated by reference in their entirety herein.

[0974] The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides areincorporated onto a solid support, or gene chip. For the purposes of thepresent invention, a peptide nucleic acid (PNA) is a polyamide type ofDNA analog and the monomeric units for adenine, guanine, thymine andcytosine are available commercially (Perceptive Biosystems). Certaincomponents of DNA, such as phosphorus, phosphorus oxides, or deoxyribosederivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A.Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,666 (1993), PNAs bind specifically and tightly to complementary DNAstrands and are not degraded by nucleases. In fact, PNA binds morestrongly to DNA than DNA itself does. This is probably because there isno electrostatic repulsion between the two strands, and also thepolyamide backbone is more flexible. Because of this, PNA/DNA duplexesbind under a wider range of stringency conditions than DNA/DNA duplexes,making it easier to perform multiplex hybridization. Smaller probes canbe used than with DNA due to the strong binding. In addition, it is morelikely that single base mismatches can be determined with PNA/DNAhybridization because a single mismatch in a PNA/DNA 15-mer lowers themelting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA15-mer duplex. Also, the absence of charge groups in PNA means thathybridization can be done at low ionic strengths and reduce possibleinterference by salt during the analysis.

[0975] The present invention is useful for detecting cancer in mammals.In particular the invention is useful during diagnosis of pathologicalcell proliferative neoplasias which include, but are not limited to:acute myelogenous leukemias including acute monocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute erythroleukemia, acute megakaryocyticleukemia, and acute undifferentiated leukemia, etc.; and chronicmyelogenous leukemias including chronic myelomonocytic leukemia, chronicgranulocytic leukemia, etc. Preferred mammals include monkeys, apes,cats, dogs, cows, pigs, horses, rabbits and humans. Particularlypreferred are humans.

[0976] Pathological cell proliferative disorders are often associatedwith inappropriate activation of proto-oncogenes. (Gelmann, E. P. etal., “The Etiology of Acute Leukemia: Molecular Genetics and ViralOncology,” in Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H.et al. eds., 161-182 (1985)). Neoplasias are now believed to result fromthe qualitative alteration of a normal cellular gene product, or fromthe quantitative modification of gene expression by insertion into thechromosome of a viral sequence, by chromosomal translocation of a geneto a more actively transcribed region, or by some other mechanism.(Gelmann et al., supra) It is likely that mutated or altered expressionof specific genes is involved in the pathogenesis of some leukemias,among other tissues and cell types. (Gelmann et al., supra) Indeed, thehuman counterparts of the oncogenes involved in some animal neoplasiashave been amplified or translocated in some cases of human leukemia andcarcinoma. (Gelmann et al., supra) For example, c-myc expression ishighly amplified in the non-lymphocytic leukemia cell line HL-60. WhenHL-60 cells are chemically induced to stop proliferation, the level ofc-myc is found to be downregulated. (International Publication Number WO91/15580) However, it has been shown that exposure of HL-60 cells to aDNA construct that is complementary to the 5′ end of c-myc or c-mybblocks translation of the corresponding mRNAs which downregulatesexpression of the c-myc or c-myb proteins and causes arrest of cellproliferation and differentiation of the treated cells. (InternationalPublication Number WO 91/15580; Wickstrom et al., Proc. Natl. Acad. Sci.85:1028 (1988); Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 (1989)).However, the skilled artisan would appreciate the present invention'susefulness would not be limited to treatment of proliferative disordersof hematopoietic cells and tissues, in light of the numerous cells andcell types of varying origins which are known to exhibit proliferativephenotypes.

[0977] In addition to the foregoing, a polynucleotide can be used tocontrol gene expression through triple helix formation or antisense DNAor RNA. Antisense techniques are discussed, for example, in Okano, J.Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Triplehelix formation is discussed in, for instance Lee et al., Nucleic AcidsResearch 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); andDervan et al., Science 251: 1360 (1991). Both methods rely on binding ofthe polynucleotide to a complementary DNA or RNA. For these techniques,preferred polynucleotides are usually oligonucleotides 20 to 40 bases inlength and complementary to either the region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat disease.

[0978] Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell.

[0979] The polynucleotides are also useful for identifying individualsfrom minute biological samples. The United States military, for example,is considering the use of restriction fragment length polymorphism(RFLP) for identification of its personnel. In this technique, anindividual's genomic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentifying personnel. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The polynucleotides of the presentinvention can be used as additional DNA markers for RFLP.

[0980] The polynucleotides of the present invention can also be used asan alternative to RFLP, by determining the actual base-by-base DNAsequence of selected portions of an individual's genome. These sequencescan be used to prepare PCR primers for amplifying and isolating suchselected DNA, which can then be sequenced. Using this technique,individuals can be identified because each individual will have a uniqueset of DNA sequences. Once an unique ID database is established for anindividual, positive identification of that individual, living or dead,can be made from extremely small tissue samples.

[0981] Forensic biology also benefits from using DNA-basedidentification techniques as disclosed herein. DNA sequences taken fromvery small biological samples such as tissues, e.g., hair or skin, orbody fluids, e.g., blood, saliva, semen, synovial fluid, amniotic fluid,breast milk, lymph, pulmonary sputum or surfactant, urine, fecal matter,etc., can be amplified using PCR. In one prior art technique, genesequences amplified from polymorphic loci, such as DQa class II HLAgene, are used in forensic biology to identify individuals. (Erlich, H.,PCR Technology, Freeman and Co. (1992).) Once these specific polymorphicloci are amplified, they are digested with one or more restrictionenzymes, yielding an identifying set of bands on a Southern blot probedwith DNA corresponding to the DQa class II HLA gene. Similarly,polynucleotides of the present invention can be used as polymorphicmarkers for forensic purposes.

[0982] There is also a need for reagents capable of identifying thesource of a particular tissue. Such need arises, for example, inforensics when presented with tissue of unknown origin. Appropriatereagents can comprise, for example, DNA probes or primers specific toparticular tissue prepared from the sequences of the present invention.Panels of such reagents can identify tissue by species and/or by organtype. In a similar fashion, these reagents can be used to screen tissuecultures for contamination.

[0983] In the very least, the polynucleotides of the present inventioncan be used as molecular weight markers on Southern gels, as diagnosticprobes for the presence of a specific mRNA in a particular cell type, asa probe to “subtract-out” known sequences in the process of discoveringnovel polynucleotides, for selecting and making oligomers for attachmentto a “gene chip” or other support, to raise anti-DNA antibodies usingDNA immunization techniques, and as an antigen to elicit an immuneresponse.

[0984] Uses of the Polypeptides

[0985] Each of the polypeptides identified herein can be used innumerous ways. The following description should be considered exemplaryand utilizes known techniques.

[0986] A polypeptide of the present invention can be used to assayprotein levels in a biological sample using antibody-based techniques.For example, protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

[0987] In addition to assaying secreted protein levels in a biologicalsample, proteins can also be detected in vivo by imaging. Antibodylabels or markers for in vivo imaging of protein include thosedetectable by X-radiography, NMR or ESR. For X-radiography, suitablelabels include radioisotopes such as barium or cesium, which emitdetectable radiation but are not overtly harmful to the subject.Suitable markers for NMR and ESR include those with a detectablecharacteristic spin, such as deuterium, which may be incorporated intothe antibody by labeling of nutrients for the relevant hybridoma.

[0988] A protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, 131I, 112In, 99 mTc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously, orintraperitoneally) into the mammal. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of 99 mTc. The labeled antibody or antibody fragment willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).)

[0989] Thus, the invention provides a diagnostic method of a disorder,which involves (a) assaying the expression of a polypeptide of thepresent invention in cells or body fluid of an individual; (b) comparingthe level of gene expression with a standard gene expression level,whereby an increase or decrease in the assayed polypeptide geneexpression level compared to the standard expression level is indicativeof a disorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0990] Moreover, polypeptides of the present invention can be used totreat disease. For example, patients can be administered a polypeptideof the present invention in an effort to replace absent or decreasedlevels of the polypeptide (e.g., insulin), to supplement absent ordecreased levels of a different polypeptide (e.g., hemoglobin S forhemoglobin B, SOD, catalase, DNA repair proteins), to inhibit theactivity of a polypeptide (e.g., an oncogene or tumor suppressor), toactivate the activity of a polypeptide (e.g., by binding to a receptor),to reduce the activity of a membrane bound receptor by competing with itfor free ligand (e.g., soluble TNF receptors used in reducinginflammation), or to bring about a desired response (e.g., blood vesselgrowth inhibition, enhancement of the immune response to proliferativecells or tissues).

[0991] Similarly, antibodies directed to a polypeptide of the presentinvention can also be used to treat disease. For example, administrationof an antibody directed to a polypeptide of the present invention canbind and reduce overproduction of the polypeptide. Similarly,administration of an antibody can activate the polypeptide, such as bybinding to a polypeptide bound to a membrane (receptor).

[0992] At the very least, the polypeptides of the present invention canbe used as molecular weight markers on SDS-PAGE gels or on molecularsieve gel filtration columns using methods well known to those of skillin the art. Polypeptides can also be used to raise antibodies, which inturn are used to measure protein expression from a recombinant cell, asa way of assessing transformation of the host cell. Moreover, thepolypeptides of the present invention can be used to test the followingbiological activities.

[0993] Gene Therapy Methods

[0994] Another aspect of the present invention is to gene therapymethods for treating disorders, diseases and conditions. The genetherapy methods relate to the introduction of nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofa polypeptide of the present invention. This method requires apolynucleotide which codes for a polypeptide of the invention thatoperatively linked to a promoter and any other genetic elementsnecessary for the expression of the polypeptide by the target tissue.Such gene therapy and delivery techniques are known in the art, see, forexample, WO90/11092, which is herein incorporated by reference.

[0995] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide of the invention ex vivo, with the engineered cells thenbeing provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, see Belldegrun et al.,J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., CancerResearch, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al.,Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38(1996)), which are herein incorporated by reference. In one embodiment,the cells which are engineered are arterial cells. The arterial cellsmay be reintroduced into the patient through direct injection to theartery, the tissues surrounding the artery, or through catheterinjection.

[0996] As discussed in more detail below, the polynucleotide constructscan be delivered by any method that delivers injectable materials to thecells of an animal, such as, injection into the interstitial space oftissues (heart, muscle, skin, lung, liver, and the like). Thepolynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

[0997] In one embodiment, the polynucleotide of the invention isdelivered as a naked polynucleotide. The term “naked” polynucleotide,DNA or RNA refers to sequences that are free from any delivery vehiclethat acts to assist, promote or facilitate entry into the cell,including viral sequences, viral particles, liposome formulations,lipofectin or precipitating agents and the like. However, thepolynucleotides of the invention can also be delivered in liposomeformulations and lipofectin formulations and the like can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference.

[0998] The polynucleotide vector constructs of the invention used in thegene therapy method are preferably constructs that will not integrateinto the host genome nor will they contain sequences that allow forreplication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia; and pEFI/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

[0999] Any strong promoter known to those skilled in the art can be usedfor driving the expression of polynucleotide sequence of the invention.Suitable promoters include adenoviral promoters, such as the adenoviralmajor late promoter; or heterologous promoters, such as thecytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV)promoter; inducible promoters, such as the MMT promoter, themetallothionein promoter; heat shock promoters; the albumin promoter;the ApoAl promoter; human globin promoters; viral thymidine kinasepromoters, such as the Herpes Simplex thymidine kinase promoter;retroviral LTRs; the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter for thepolynucleotides of the invention.

[1000] Unlike other gene therapy techniques, one major advantage ofintroducing naked nucleic acid sequences into target cells is thetransitory nature of the polynucleotide synthesis in the cells. Studieshave shown that non-replicating DNA sequences can be introduced intocells to provide production of the desired polypeptide for periods of upto six months.

[1001] The polynucleotide construct of the invention can be delivered tothe interstitial space of tissues within the an animal, including ofmuscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart,lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

[1002] For the naked nucleic acid sequence injection, an effectivedosage amount of DNA or RNA will be in the range of from about 0.05mg/kg body weight to about 50 mg/kg body weight. Preferably the dosagewill be from about 0.005 mg/kg to about 20 mg/kg and more preferablyfrom about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan ofordinary skill will appreciate, this dosage will vary according to thetissue site of injection. The appropriate and effective dosage ofnucleic acid sequence can readily be determined by those of ordinaryskill in the art and may depend on the condition being treated and theroute of administration.

[1003] The preferred route of administration is by the parenteral routeof injection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

[1004] The naked polynucleotides are delivered by any method known inthe art, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, and so-called “gene guns”. These delivery methods are known inthe art.

[1005] The constructs may also be delivered with delivery vehicles suchas viral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

[1006] In certain embodiments, the polynucleotide constructs of theinvention are complexed in a liposome preparation. Liposomalpreparations for use in the instant invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations. However, cationic liposomes are particularly preferredbecause a tight charge complex can be formed between the cationicliposome and the polyanionic nucleic acid. Cationic liposomes have beenshown to mediate intracellular delivery of plasmid DNA (Felgner et al.,Proc. Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is hereinincorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci.USA, 86:6077-6081 (1989), which is herein incorporated by reference);and purified transcription factors (Debs et al., J. Biol. Chem.,265:10189-10192 (1990), which is herein incorporated by reference), infunctional form.

[1007] Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

[1008] Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication NO: WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., Feigner etal., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

[1009] Similarly, anionic and neutral liposomes are readily available,such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easilyprepared using readily available materials. Such materials includephosphatidyl, choline, cholesterol, phosphatidyl ethanolamine,dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol(DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. Thesematerials can also be mixed with the DOTMA and DOTAP starting materialsin appropriate ratios. Methods for making liposomes using thesematerials are well known in the art.

[1010] For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

[1011] The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology, 101:512-527 (1983), which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca 2-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilsonet al., Cell, 17:77 (1979)); ether injection (Deamer et al., Biochem.Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res.Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA,76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad.Sci. USA, 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley etal., J. Biol. Chem., 255:10431 (1980); Szoka et al., Proc. Natl. Acad.Sci. USA, 75:145 (1978); Schaefer-Ridder et al., Science, 215:166(1982)), which are herein incorporated by reference.

[1012] Generally, the ratio of DNA to liposomes will be from about 10:1to about 1:10. Preferably, the ration will be from about 5:1 to about1:5. More preferably, the ration will be about 3:1 to about 1:3. Stillmore preferably, the ratio will be about 1:1.

[1013] U.S. Pat. No. 5,676,954 (which is herein incorporated byreference) reports on the injection of genetic material, complexed withcationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355,4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication NO: WO 94/9469 (which areherein incorporated by reference) provide cationic lipids for use intransfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466,5,693,622, 5,580,859, 5,703,055, and international publication NO: WO94/9469 (which are herein incorporated by reference) provide methods fordelivering DNA-cationic lipid complexes to mammals.

[1014] In certain embodiments, cells are engineered, ex vivo or in vivo,using a retroviral particle containing RNA which comprises a sequenceencoding polypeptides of the invention. Retroviruses from which theretroviral plasmid vectors may be derived include, but are not limitedto, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus,and mammary tumor virus.

[1015] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, R-2, R-AM, PA12, T19-14×, VT-19-17-H2, RCRE, RCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy, 1:5-14 (1990), which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

[1016] The producer cell line generates infectious retroviral vectorparticles which include polynucleotide encoding polypeptides of theinvention. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express polypeptides of the invention.

[1017] In certain other embodiments, cells are engineered, ex vivo or invivo, with polynucleotides of the invention contained in an adenovirusvector. Adenovirus can be manipulated such that it encodes and expressespolypeptides of the invention, and at the same time is inactivated interms of its ability to replicate in a normal lytic viral life cycle.Adenovirus expression is achieved without integration of the viral DNAinto the host cell chromosome, thereby alleviating concerns aboutinsertional mutagenesis. Furthermore, adenoviruses have been used aslive enteric vaccines for many years with an excellent safety profile(Schwartz et al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally,adenovirus mediated gene transfer has been demonstrated in a number ofinstances including transfer of alpha-1-antitrypsin and CFFR to thelungs of cotton rats (Rosenfeld et al., Science, 252:431-434 (1991);Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA,76:6606 (1979)).

[1018] Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel., 3:499-503 (1993); Rosenfeld et al., Cell, 68:143-155 (1992);Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al.,Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

[1019] Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

[1020] In certain other embodiments, the cells are engineered, ex vivoor in vivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol.,158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

[1021] For example, an appropriate AAV vector for use in the presentinvention will include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructcontaining polynucleotides of the invention is inserted into the AAVvector using standard cloning methods, such as those found in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press(1989). The recombinant AAV vector is then transfected into packagingcells which are infected with a helper virus, using any standardtechnique, including lipofection, electroporation, calcium phosphateprecipitation, etc. Appropriate helper viruses include adenoviruses,cytomegaloviruses, vaccinia viruses, or herpes viruses. Once thepackaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct of the invention. These viral particles are then used totransduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the polynucleotide construct integrated into itsgenome, and will express the desired gene product.

[1022] Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding the polypeptide sequence of interest) via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot normally expressed in the cells, or is expressed at a lower levelthan desired.

[1023] Polynucleotide constructs are made, using standard techniquesknown in the art, which contain the promoter with targeting sequencesflanking the promoter. Suitable promoters are described herein. Thetargeting sequence is sufficiently complementary to an endogenoussequence to permit homologous recombination of the promoter-targetingsequence with the endogenous sequence. The targeting sequence will besufficiently near the 5′ end of the desired endogenous polynucleotidesequence so the promoter will be operably linked to the endogenoussequence upon homologous recombination.

[1024] The promoter and the targeting sequences can be amplified usingPCR. Preferably, the amplified promoter contains distinct restrictionenzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

[1025] The promoter-targeting sequence construct is delivered to thecells, either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

[1026] The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence.

[1027] The polynucleotides encoding polypeptides of the presentinvention may be administered along with other polynucleotides encodingother angiogenic proteins. Angiogenic proteins include, but are notlimited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2(VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor alpha and beta,platelet-derived endothelial cell growth factor, platelet-derived growthfactor, tumor necrosis factor alpha, hepatocyte growth factor, insulinlike growth factor, colony stimulating factor, macrophage colonystimulating factor, granulocyte/macrophage colony stimulating factor,and nitric oxide synthase.

[1028] Preferably, the polynucleotide encoding a polypeptide of theinvention contains a secretory signal sequence that facilitatessecretion of the protein. Typically, the signal sequence is positionedin the coding region of the polynucleotide to be expressed towards or atthe 5′ end of the coding region. The signal sequence may be homologousor heterologous to the polynucleotide of interest and may be homologousor heterologous to the cells to be transfected. Additionally, the signalsequence may be chemically synthesized using methods known in the art.

[1029] Any mode of administration of any of the above-describedpolynucleotides constructs can be used so long as the mode results inthe expression of one or more molecules in an amount sufficient toprovide a therapeutic effect. This includes direct needle injection,systemic injection, catheter infusion, biolistic injectors, particleaccelerators (i.e., “gene guns”), gelfoam sponge depots, othercommercially available depot materials, osmotic pumps (e.g., Alzaminipumps), oral or suppositorial solid (tablet or pill) pharmaceuticalformulations, and decanting or topical applications during surgery. Forexample, direct injection of naked calcium phosphate-precipitatedplasmid into rat liver and rat spleen or a protein-coated plasmid intothe portal vein has resulted in gene expression of the foreign gene inthe rat livers. (Kaneda et al., Science, 243:375 (1989)).

[1030] A preferred method of local administration is by directinjection. Preferably, a recombinant molecule of the present inventioncomplexed with a delivery vehicle is administered by direct injectioninto or locally within the area of arteries. Administration of acomposition locally within the area of arteries refers to injecting thecomposition centimeters and preferably, millimeters within arteries.

[1031] Another method of local administration is to contact apolynucleotide construct of the present invention in or around asurgical wound. For example, a patient can undergo surgery and thepolynucleotide construct can be coated on the surface of tissue insidethe wound or the construct can be injected into areas of tissue insidethe wound.

[1032] Therapeutic compositions useful in systemic administration,include recombinant molecules of the present invention complexed to atargeted delivery vehicle of the present invention. Suitable deliveryvehicles for use with systemic administration comprise liposomescomprising ligands for targeting the vehicle to a particular site.

[1033] Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA,189:11277-11281(1992), which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

[1034] Determining an effective amount of substance to be delivered candepend upon a number of factors including, for example, the chemicalstructure and biological activity of the substance, the age and weightof the animal, the precise condition requiring treatment and itsseverity, and the route of administration. The frequency of treatmentsdepends upon a number of factors, such as the amount of polynucleotideconstructs administered per dose, as well as the health and history ofthe subject. The precise amount, number of doses, and timing of doseswill be determined by the attending physician or veterinarian.Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs,with humans being particularly

[1035] Biological Activities

[1036] The polynucleotides or polypeptides, or agonists or antagonistsof the present invention can be used in assays to test for one or morebiological activities. If these polynucleotides and polypeptides doexhibit activity in a particular assay, it is likely that thesemolecules may be involved in the diseases associated with the biologicalactivity. Thus, the polynucleotides or polypeptides, or agonists orantagonists could be used to treat the associated disease.

[1037] Immune Activity

[1038] The polynucleotides or polypeptides, or agonists or antagonistsof the present invention may be useful in treating deficiencies ordisorders of the immune system, by activating or inhibiting theproliferation, differentiation, or mobilization (chemotaxis) of immunecells. Immune cells develop through a process called hematopoiesis,producing myeloid (platelets, red blood cells, neutrophils, andmacrophages) and lymphoid (B and T lymphocytes) cells from pluripotentstem cells. The etiology of these immune deficiencies or disorders maybe genetic, somatic, such as cancer or some autoimmune disorders,acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used as a marker or detector of a particularimmune system disease or disorder.

[1039] A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may be useful in treating or detectingdeficiencies or disorders of hematopoietic cells. A polynucleotides orpolypeptides, or agonists or antagonists of the present invention couldbe used to increase differentiation and proliferation of hematopoieticcells, including the pluripotent stem cells, in an effort to treat thosedisorders associated with a decrease in certain (or many) typeshematopoietic cells. Examples of immunologic deficiency syndromesinclude, but are not limited to: blood protein disorders (e.g.agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, commonvariable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLVinfection, leukocyte adhesion deficiency syndrome, lymphopenia,phagocyte bactericidal dysfunction, severe combined immunodeficiency(SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, orhemoglobinuria.

[1040] Moreover, a polynucleotides or polypeptides, or agonists orantagonists of the present invention could also be used to modulatehemostatic (the stopping of bleeding) or thrombolytic activity (clotformation). For example, by increasing hemostatic or thrombolyticactivity, a polynucleotides or polypeptides, or agonists or antagonistsof the present invention could be used to treat blood coagulationdisorders (e.g., afibrinogenemia, factor deficiencies), blood plateletdisorders (e.g. thrombocytopenia), or wounds resulting from trauma,surgery, or other causes. Alternatively, a polynucleotides orpolypeptides, or agonists or antagonists of the present invention thatcan decrease hemostatic or thrombolytic activity could be used toinhibit or dissolve clotting. These molecules could be important in thetreatment of heart attacks (infarction), strokes, or scarring.

[1041] A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may also be useful in treating or detectingautoimmune disorders. Many autoimmune disorders result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Therefore, the administration of apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention that inhibits an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune disorders.

[1042] Examples of autoimmune disorders that can be treated or detectedby the present invention include, but are not limited to: Addison'sDisease, hemolytic anemia, antiphospholipid syndrome, rheumatoidarthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis,Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, MyastheniaGravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Thyroiditis, Systemic Lupus Erythematosus, AutoimmunePulmonary Inflammation, Guillain-Barre Syndrome, insulin dependentdiabetes mellitis, and autoimmune inflammatory eye disease.

[1043] Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by a polynucleotides or polypeptides, or agonists orantagonists of the present invention. Moreover, these molecules can beused to treat anaphylaxis, hypersensitivity to an antigenic molecule, orblood group incompatibility.

[1044] A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may also be used to treat and/or prevent organrejection or graft-versus-host disease (GVHD). Organ rejection occurs byhost immune cell destruction of the transplanted tissue through animmune response. Similarly, an immune response is also involved in GVHD,but, in this case, the foreign transplanted immune cells destroy thehost tissues. The administration of a polynucleotides or polypeptides,or agonists or antagonists of the present invention that inhibits animmune response, particularly the proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventing organrejection or GVHD.

[1045] Similarly, a polynucleotides or polypeptides, or agonists orantagonists of the present invention may also be used to modulateinflammation. For example, the polypeptide or polynucleotide or agonistsor antagonist may inhibit the proliferation and differentiation of cellsinvolved in an inflammatory response. These molecules can be used totreat inflammatory conditions, both chronic and acute conditions,including inflammation associated with infection (e.g., septic shock,sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

[1046] Hyperproliferative Disorders

[1047] A polynucleotides or polypeptides, or agonists or antagonists ofthe invention can be used to treat or detect hyperproliferativedisorders, including neoplasms. A polynucleotides or polypeptides, oragonists or antagonists of the present invention may inhibit theproliferation of the disorder through direct or indirect interactions.Alternatively, a polynucleotides or polypeptides, or agonists orantagonists of the present invention may proliferate other cells whichcan inhibit the hyperproliferative disorder.

[1048] For example, by increasing an immune response, particularlyincreasing antigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

[1049] Examples of hyperproliferative disorders that can be treated ordetected by a polynucleotides or polypeptides, or agonists orantagonists of the present invention include, but are not limited toneoplasms located in the: abdomen, bone, breast, digestive system,liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid,pituitary, testicles, ovary, thymus, thyroid), eye, head and neck,nervous (central and peripheral), lymphatic system, pelvic, skin, softtissue, spleen, thoracic, and urogenital.

[1050] Similarly, other hyperproliferative disorders can also be treatedor detected by a polynucleotides or polypeptides, or agonists orantagonists of the present invention. Examples of suchhyperproliferative disorders include, but are not limited to:hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia,Gaucher's Disease, histiocytosis, and any other hyperproliferativedisease, besides neoplasia, located in an organ system listed above.

[1051] One preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

[1052] Thus, the present invention provides a method for treating cellproliferative disorders by inserting into an abnormally proliferatingcell a polynucleotide of the present invention, wherein saidpolynucleotide represses said expression.

[1053] Another embodiment of the present invention provides a method oftreating cell-proliferative disorders in individuals comprisingadministration of one or more active gene copies of the presentinvention to an abnormally proliferating cell or cells. In a preferredembodiment, polynucleotides of the present invention is a DNA constructcomprising a recombinant expression vector effective in expressing a DNAsequence encoding said polynucleotides. In another preferred embodimentof the present invention, the DNA construct encoding the poynucleotidesof the present invention is inserted into cells to be treated utilizinga retrovirus, or more preferably an adenoviral vector (See G J. Nabel,et. al., PNAS 1999 96: 324-326, which is hereby incorporated byreference). In a most preferred embodiment, the viral vector isdefective and will not transform non-proliferating cells, onlyproliferating cells. Moreover, in a preferred embodiment, thepolynucleotides of the present invention inserted into proliferatingcells either alone, or in combination with or fused to otherpolynucleotides, can then be modulated via an external stimulus (i.e.magnetic, specific small molecule, chemical, or drug administration,etc.), which acts upon the promoter upstream of said polynucleotides toinduce expression of the encoded protein product. As such the beneficialtherapeutic affect of the present invention may be expressly modulated(i.e. to increase, decrease, or inhibit expression of the presentinvention) based upon said external stimulus.

[1054] Polynucleotides of the present invention may be useful inrepressing expression of oncogenic genes or antigens. By “repressingexpression of the oncogenic genes” is intended the suppression of thetranscription of the gene, the degradation of the gene transcript(pre-message RNA), the inhibition of splicing, the destruction of themessenger RNA, the prevention of the post-translational modifications ofthe protein, the destruction of the protein, or the inhibition of thenormal function of the protein.

[1055] For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

[1056] The polynucleotides of the present invention may be delivereddirectly to cell proliferative disorder/disease sites in internalorgans, body cavities and the like by use of imaging devices used toguide an injecting needle directly to the disease site. Thepolynucleotides of the present invention may also be administered todisease sites at the time of surgical intervention.

[1057] By “cell proliferative disease” is meant any human or animaldisease or disorder, affecting any one or any combination of organs,cavities, or body parts, which is characterized by single or multiplelocal abnormal proliferations of cells, groups of cells, or tissues,whether benign or malignant.

[1058] Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

[1059] The present invention is further directed to antibody-basedtherapies which involve administering of anti-polypeptides andanti-polynucleotide antibodies to a mammalian, preferably human, patientfor treating one or more of the described disorders. Methods forproducing anti-polypeptides and anti-polynucleotide antibodiespolyclonal and monoclonal antibodies are described in detail elsewhereherein. Such antibodies may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein.

[1060] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[1061] In particular, the antibodies, fragments and derivatives of thepresent invention are useful for treating a subject having or developingcell proliferative and/or differentiation disorders as described herein.Such treatment comprises administering a single or multiple doses of theantibody, or a fragment, derivative, or a conjugate thereof.

[1062] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example, which serve toincrease the number or activity of effector cells which interact withthe antibodies.

[1063] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides,including fragments thereof. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M,10⁻⁷M, 5×10⁻⁸ M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10¹¹ M,10⁻¹¹M, 5×10¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M,and 10⁻¹⁵M.

[1064] Moreover, polypeptides of the present invention are useful ininhibiting the angiogenesis of proliferative cells or tissues, eitheralone, as a protein fusion, or in combination with other polypeptidesdirectly or indirectly, as described elsewhere herein. In a mostpreferred embodiment, said anti-angiogenesis effect may be achievedindirectly, for example, through the inhibition of hematopoietic,tumor-specific cells, such as tumor-associated macrophages (See JosephIB, et al. J Natl Cancer Inst, 90(21): 1648-53 (1998), which is herebyincorporated by reference). Antibodies directed to polypeptides orpolynucleotides of the present invention may also result in inhibitionof angiogenesis directly, or indirectly (See Witte L, et al., CancerMetastasis Rev. 17(2):155-61 (1998), which is hereby incorporated byreference)).

[1065] Polypeptides, including protein fusions, of the presentinvention, or fragments thereof may be useful in inhibitingproliferative cells or tissues through the induction of apoptosis. Saidpolypeptides may act either directly, or indirectly to induce apoptosisof proliferative cells and tissues, for example in the activation of adeath-domain receptor, such as tumor necrosis factor (TNF) receptor-1,CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein(TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and−2 (See Schulze-Osthoff K, et. al., Eur J Biochem 254(3):439-59 (1998),which is hereby incorporated by reference). Moreover, in anotherpreferred embodiment of the present invention, said polypeptides mayinduce apoptosis through other mechanisms, such as in the activation ofother proteins which will activate apoptosis, or through stimulating theexpression of said proteins, either alone or in combination with smallmolecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins,antiinflammatory proteins (See for example, Mutat Res 400(1-2):447-55(1998), Med Hypotheses.50(5):423-33 (1998), Chem Biol Interact. Apr24;111-112:23-34 (1998), J Mol Med.76(6):402-12 (1998), Int J TissueReact;20(1):3-15 (1998), which are all hereby incorporated byreference).

[1066] Polypeptides, including protein fusions to, or fragments thereof,of the present invention are useful in inhibiting the metastasis ofproliferative cells or tissues. Inhibition may occur as a direct resultof administering polypeptides, or antibodies directed to saidpolypeptides as described elsewhere herein, or indirectly, such asactivating the expression of proteins known to inhibit metastasis, forexample alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol1998;231:125-41, which is hereby incorporated by reference). Suchtherapeutic affects of the present invention may be achieved eitheralone, or in combination with small molecule drugs or adjuvants.

[1067] In another embodiment, the invention provides a method ofdelivering compositions containing the polypeptides of the invention(e.g., compositions containing polypeptides or polypeptide antibodiesassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs) to targeted cells expressing the polypeptide of thepresent invention. Polypeptides or polypeptide antibodies of theinvention may be associated with heterologous polypeptides, heterologousnucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionicand/or covalent interactions.

[1068] Polypeptides, protein fusions to, or fragments thereof, of thepresent invention are useful in enhancing the immunogenicity and/orantigenicity of proliferating cells or tissues, either directly, such aswould occur if the polypeptides of the present invention ‘vaccinated’the immune response to respond to proliferative antigens and immunogens,or indirectly, such as in activating the expression of proteins known toenhance the immune response (e.g. chemokines), to said antigens andimmunogens.

[1069] Cardiovascular Disorders

[1070] Polynucleotides or polypeptides, or agonists or antagonists ofthe invention may be used to treat cardiovascular disorders, includingperipheral artery disease, such as limb ischemia.

[1071] Cardiovascular disorders include cardiovascular abnormalities,such as arterio-arterial fistula, arteriovenous fistula, cerebralarteriovenous malformations, congenital heart defects, pulmonaryatresia, and Scimitar Syndrome. Congenital heart defects include aorticcoarctation, cor triatriatum, coronary vessel anomalies, crisscrossheart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,Eisenmenger complex, hypoplastic left heart syndrome, levocardia,tetralogy of fallot, transposition of great vessels, double outlet rightventricle, tricuspid atresia, persistent truncus arteriosus, and heartseptal defects, such as aortopulmonary septal defect, endocardialcushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricularheart septal defects.

[1072] Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

[1073] Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

[1074] Heart valve disease include aortic valve insufficiency, aorticvalve stenosis, hear murmurs, aortic valve prolapse, mitral valveprolapse, tricuspid valve prolapse, mitral valve insufficiency, mitralvalve stenosis, pulmonary atresia, pulmonary valve insufficiency,pulmonary valve stenosis, tricuspid atresia, tricuspid valveinsufficiency, and tricuspid valve stenosis.

[1075] Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic, subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

[1076] Myocardial ischemias include coronary disease, such as anginapectoris, coronary aneurysm, coronary arteriosclerosis, coronarythrombosis, coronary vasospasm, myocardial infarction and myocardialstunning.

[1077] Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

[1078] Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

[1079] Arterial occlusive diseases include arteriosclerosis,intermittent claudication, carotid stenosis, fibromuscular dysplasias,mesenteric vascular occlusion, Moyamoya disease, renal arteryobstruction, retinal artery occlusion, and thromboangiitis obliterans.

[1080] Cerebrovascular disorders include carotid artery diseases,cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebralartery diseases, cerebral embolism and thrombosis, carotid arterythrombosis, sinus thrombosis, Wallenberg's syndrome, cerebralhemorrhage, epidural hematoma, subdural hematoma, subaraxhnoidhemorrhage, cerebral infarction, cerebral ischemia (includingtransient), subclavian steal syndrome, periventricular leukomalacia,vascular headache, cluster headache, migraine, and vertebrobasilarinsufficiency.

[1081] Embolisms include air embolisms, amniotic fluid embolisms,cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonaryembolisms, and thromoboembolisms. Thrombosis include coronarythrombosis, hepatic vein thrombosis, retinal vein occlusion, carotidartery thrombosis, sinus thrombosis, Wallenberg's syndrome, andthrombophlebitis.

[1082] Ischemia includes cerebral ischemia, ischemic colitis,compartment syndromes, anterior compartment syndrome, myocardialischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitisincludes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

[1083] Polynucleotides or polypeptides, or agonists or antagonists ofthe invention, are especially effective for the treatment of criticallimb ischemia and coronary disease.

[1084] Polypeptides may be administered using any method known in theart, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, biolistic injectors, particle accelerators, gelfoam spongedepots, other commercially available depot materials, osmotic pumps,oral or suppositorial solid pharmaceutical formulations, decanting ortopical applications during surgery, aerosol delivery. Such methods areknown in the art. Polypeptides of the invention may be administered aspart of a Therapeutic, described in more detail below. Methods ofdelivering polynucleotides of the invention are described in more detailherein.

[1085] Anti-Angiogenesis Activity

[1086] The naturally occurring balance between endogenous stimulatorsand inhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442447 (1987).

[1087] The present invention provides for treatment of diseases ordisorders associated with neovascularization by administration of thepolynucleotides and/or polypeptides of the invention, as well asagonists or antagonists of the present invention. Malignant andmetastatic conditions which can be treated with the polynucleotides andpolypeptides, or agonists or antagonists of the invention include, butare not limited to, malignancies, solid tumors, and cancers describedherein and otherwise known in the art (for a review of such disorders,see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia(1985)). Thus, the present invention provides a method of treating anangiogenesis-related disease and/or disorder, comprising administeringto an individual in need thereof a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist of the invention.For example, polynucleotides, polypeptides, antagonists and/or agonistsmay be utilized in a variety of additional methods in order totherapeutically treat a cancer or tumor. Cancers which may be treatedwith polynucleotides, polypeptides, antagonists and/or agonists include,but are not limited to solid tumors, including prostate, lung, breast,ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid,biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,bladder, thyroid cancer; primary tumors and metastases; melanomas;glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lungcancer; colorectal cancer; advanced malignancies; and blood bom tumorssuch as leukemias. For example, polynucleotides, polypeptides,antagonists and/or agonists may be delivered topically, in order totreat cancers such as skin cancer, head and neck tumors, breast tumors,and Kaposi's sarcoma.

[1088] Within yet other aspects, polynucleotides, polypeptides,antagonists and/or agonists may be utilized to treat superficial formsof bladder cancer by, for example, intravesical administration.Polynucleotides, polypeptides, antagonists and/or agonists may bedelivered directly into the tumor, or near the tumor site, via injectionor a catheter. Of course, as the artisan of ordinary skill willappreciate, the appropriate mode of administration will vary accordingto the cancer to be treated. Other modes of delivery are discussedherein.

[1089] Polynucleotides, polypeptides, antagonists and/or agonists may beuseful in treating other disorders, besides cancers, which involveangiogenesis. These disorders include, but are not limited to: benigntumors, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas; artheroscleric plaques; ocularangiogenic diseases, for example, diabetic retinopathy, retinopathy ofprematurity, macular degeneration, corneal graft rejection, neovascularglaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis andPterygia (abnormal blood vessel growth) of the eye; rheumatoidarthritis; psoriasis; delayed wound healing; endometriosis;vasculogenesis; granulations; hypertrophic scars (keloids); nonunionfractures; scleroderma; trachoma; vascular adhesions; myocardialangiogenesis; coronary collaterals; cerebral collaterals; arteriovenousmalformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;fibromuscular dysplasia; wound granulation; Crohn's disease; andatherosclerosis.

[1090] For example, within one aspect of the present invention methodsare provided for treating hypertrophic scars and keloids, comprising thestep of administering a polynucleotide, polypeptide, antagonist and/oragonist of the invention to a hypertrophic scar or keloid.

[1091] Within one embodiment of the present invention polynucleotides,polypeptides, antagonists and/or agonists are directly injected into ahypertrophic scar or keloid, in order to prevent the progression ofthese lesions. This therapy is of particular value in the prophylactictreatment of conditions which are known to result in the development ofhypertrophic scars and keloids (e.g., burns), and is preferablyinitiated after the proliferative phase has had time to progress(approximately 14 days after the initial injury), but beforehypertrophic scar or keloid development. As noted above, the presentinvention also provides methods for treating neovascular diseases of theeye, including for example, comeal neovascularization, neovascularglaucoma, proliferative diabetic retinopathy, retrolental fibroplasiaand macular degeneration.

[1092] Moreover, Ocular disorders associated with neovascularizationwhich can be treated with the polynucleotides and polypeptides of thepresent invention (including agonists and/or antagonists) include, butare not limited to: neovascular glaucoma, diabetic retinopathy,retinoblastoma, retrolental fibroplasia, uveitis, retinopathy ofprematurity macular degeneration, corneal graft neovascularization, aswell as other eye inflammatory diseases, ocular tumors and diseasesassociated with choroidal or iris neovascularization. See, e.g., reviewsby Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al.,Surv. Ophthal. 22:291-312 (1978).

[1093] Thus, within one aspect of the present invention methods areprovided for treating neovascular diseases of the eye such as cornealneovascularization (including corneal graft neovascularization),comprising the step of administering to a patient a therapeuticallyeffective amount of a compound (as described above) to the cornea, suchthat the formation of blood vessels is inhibited. Briefly, the cornea isa tissue which normally lacks blood vessels. In certain pathologicalconditions however, capillaries may extend into the cornea from thepericorneal vascular plexus of the limbus. When the cornea becomesvascularized, it also becomes clouded, resulting in a decline in thepatient's visual acuity. Visual loss may become complete if the corneacompletely opacitates. A wide variety of disorders can result in cornealneovascularization, including for example, corneal infections (e.g.,trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis),immunological processes (e.g., graft rejection and Stevens-Johnson'ssyndrome), alkali burns, trauma, inflammation (of any cause), toxic andnutritional deficiency states, and as a complication of wearing contactlenses.

[1094] Within particularly preferred embodiments of the invention, maybe prepared for topical administration in saline (combined with any ofthe preservatives and antimicrobial agents commonly used in ocularpreparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily. Alternatively, anti-angiogenic compositions, prepared asdescribed above, may also be administered directly to the cornea. Withinpreferred embodiments, the anti-angiogenic composition is prepared witha muco-adhesive polymer which binds to cornea. Within furtherembodiments, the anti-angiogenic factors or anti-angiogenic compositionsmay be utilized as an adjunct to conventional steroid therapy. Topicaltherapy may also be useful prophylactically in comeal lesions which areknown to have a high probability of inducing an angiogenic response(such as chemical burns). In these instances the treatment, likely incombination with steroids, may be instituted immediately to help preventsubsequent complications.

[1095] Within other embodiments, the compounds described above may beinjected directly into the corneal stroma by an ophthalmologist undermicroscopic guidance. The preferred site of injection may vary with themorphology of the individual lesion, but the goal of the administrationwould be to place the composition at the advancing front of thevasculature (i.e., interspersed between the blood vessels and the normalcornea). In most cases this would involve perilimbic corneal injectionto “protect” the cornea from the advancing blood vessels. This methodmay also be utilized shortly after a corneal insult in order toprophylactically prevent comeal neovascularization. In this situationthe material could be injected in the perilimbic cornea interspersedbetween the corneal lesion and its undesired potential limbic bloodsupply. Such methods may also be utilized in a similar fashion toprevent capillary invasion of transplanted corneas. In asustained-release form injections might only be required 2-3 times peryear. A steroid could also be added to the injection solution to reduceinflammation resulting from the injection itself.

[1096] Within another aspect of the present invention, methods areprovided for treating neovascular glaucoma, comprising the step ofadministering to a patient a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist to the eye, suchthat the formation of blood vessels is inhibited. In one embodiment, thecompound may be administered topically to the eye in order to treatearly forms of neovascular glaucoma. Within other embodiments, thecompound may be implanted by injection into the region of the anteriorchamber angle. Within other embodiments, the compound may also be placedin any location such that the compound is continuously released into theaqueous humor. Within another aspect of the present invention, methodsare provided for treating proliferative diabetic retinopathy, comprisingthe step of administering to a patient a therapeutically effectiveamount of a polynucleotide, polypeptide, antagonist and/or agonist tothe eyes, such that the formation of blood vessels is inhibited.

[1097] Within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection into theaqueous humor or the vitreous, in order to increase the localconcentration of the polynucleotide, polypeptide, antagonist and/oragonist in the retina. Preferably, this treatment should be initiatedprior to the acquisition of severe disease requiring photocoagulation.

[1098] Within another aspect of the present invention, methods areprovided for treating retrolental fibroplasia, comprising the step ofadministering to a patient a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist to the eye, suchthat the formation of blood vessels is inhibited. The compound may beadministered topically, via intravenous injection and/or via intraocularimplants.

[1099] Additionally, disorders which can be treated with thepolynucleotides, polypeptides, agonists and/or agonists include, but arenot limited to, hemangioma, arthritis, psoriasis, angiofibroma,atherosclerotic plaques, delayed wound healing, granulations, hemophilicjoints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome,pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

[1100] Moreover, disorders and/or states, which can be treated with betreated with the polynucleotides, polypeptides, agonists and/or agonistsinclude, but are not limited to, solid tumors, blood born tumors such asleukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, forexample hemangiomas, acoustic neuromas, neurofibromas, trachomas, andpyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenicdiseases, for example, diabetic retinopathy, retinopathy of prematurity,macular degeneration, corneal graft rejection, neovascular glaucoma,retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayedwound healing, endometriosis, vascluogenesis, granulations, hypertrophicscars (keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

[1101] In one aspect of the birth control method, an amount of thecompound sufficient to block embryo implantation is administered beforeor after intercourse and fertilization have occurred, thus providing aneffective method of birth control, possibly a “morning after” method.Polynucleotides, polypeptides, agonists and/or agonists may also be usedin controlling menstruation or administered as either a peritoneallavage fluid or for peritoneal implantation in the treatment ofendometriosis.

[1102] Polynucleotides, polypeptides, agonists and/or agonists of thepresent invention may be incorporated into surgical sutures in order toprevent stitch granulomas.

[1103] Polynucleotides, polypeptides, agonists and/or agonists may beutilized in a wide variety of surgical procedures. For example, withinone aspect of the present invention a compositions (in the form of, forexample, a spray or film) may be utilized to coat or spray an area priorto removal of a tumor, in order to isolate normal surrounding tissuesfrom malignant tissue, and/or to prevent the spread of disease tosurrounding tissues. Within other aspects of the present invention,compositions (e.g., in the form of a spray) may be delivered viaendoscopic procedures in order to coat tumors, or inhibit angiogenesisin a desired locale. Within yet other aspects of the present invention,surgical meshes which have been coated with anti-angiogenic compositionsof the present invention may be utilized in any procedure wherein asurgical mesh might be utilized. For example, within one embodiment ofthe invention a surgical mesh laden with an anti-angiogenic compositionmay be utilized during abdominal cancer resection surgery (e.g.,subsequent to colon resection) in order to provide support to thestructure, and to release an amount of the anti-angiogenic factor.

[1104] Within further aspects of the present invention, methods areprovided for treating tumor excision sites, comprising administering apolynucleotide, polypeptide, agonist and/or agonist to the resectionmargins of a tumor subsequent to excision, such that the localrecurrence of cancer and the formation of new blood vessels at the siteis inhibited. Within one embodiment of the invention, theanti-angiogenic compound is administered directly to the tumor excisionsite (e.g., applied by swabbing, brushing or otherwise coating theresection margins of the tumor with the anti-angiogenic compound).Alternatively, the anti-angiogenic compounds may be incorporated intoknown surgical pastes prior to administration. Within particularlypreferred embodiments of the invention, the anti-angiogenic compoundsare applied after hepatic resections for malignancy, and afterneurosurgical operations.

[1105] Within one aspect of the present invention, polynucleotides,polypeptides, agonists and/or agonists may be administered to theresection margin of a wide variety of tumors, including for example,breast, colon, brain and hepatic tumors. For example, within oneembodiment of the invention, anti-angiogenic compounds may beadministered to the site of a neurological tumor subsequent to excision,such that the formation of new blood vessels at the site are inhibited.

[1106] The polynucleotides, polypeptides, agonists and/or agonists ofthe present invention may also be administered along with otheranti-angiogenic factors. Representative examples of otheranti-angiogenic factors include: Anti-Invasive Factor, retinoic acid andderivatives thereof, paclitaxel, Suramin, Tissue Inhibitor ofMetalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2,and various forms of the lighter “d group” transition metals.

[1107] Lighter “d group” transition metals include, for example,vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species.Such transition metal species may form transition metal complexes.Suitable complexes of the above-mentioned transition metal speciesinclude oxo transition metal complexes.

[1108] Representative examples of vanadium complexes include oxovanadium complexes such as vanadate and vanadyl complexes. Suitablevanadate complexes include metavanadate and orthovanadate complexes suchas, for example, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

[1109] Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

[1110] A wide variety of other anti-angiogenic factors may also beutilized within the context of the present invention. Representativeexamples include platelet factor 4; protamine sulphate; sulphated chitinderivatives (prepared from queen crab shells), (Murata et al., CancerRes. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex(SP-PG) (the function of this compound may be enhanced by the presenceof steroids such as estrogen, and tamoxifen citrate); Staurosporine;modulators of matrix metabolism, including for example, proline analogs,cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4): 1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;Angiostatic steroid; AGM-1470; carboxynaminolmidazole; andmetalloproteinase inhibitors such as BB94.

[1111] Diseases at the Cellular Level

[1112] Diseases associated with increased cell survival or theinhibition of apoptosis that could be treated or detected by thepolynucleotides or polypeptides and/or antagonists or agonists of theinvention, include cancers (such as follicular lymphomas, carcinomaswith p53 mutations, and hormone-dependent tumors, including, but notlimited to colon cancer, cardiac tumors, pancreatic cancer, melanoma,retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicularcancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection. In preferred embodiments, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention are used to inhibit growth, progression, and/or metastasis ofcancers, in particular those listed above.

[1113] Additional diseases or conditions associated with increased cellsurvival that could be treated or detected by the polynucleotides orpolypeptides, or agonists or antagonists of the invention, include, butare not limited to, progression, and/or metastases of malignancies andrelated disorders such as leukemia (including acute leukemias (e.g.,acute lymphocytic leukemia, acute myelocytic leukemia (includingmyeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

[1114] Diseases associated with increased apoptosis that could betreated or detected by the polynucleotides or polypeptides, and/oragonists or antagonists of the invention, include AIDS;neurodegenerative disorders (such as Alzheimer's disease, Parkinson'sdisease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellardegeneration and brain tumor or prior associated disease); autoimmunedisorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (e.g., hepatitis related liver injury, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, cachexiaand anorexia.

[1115] Wound Healing and Epithelial Cell Proliferation

[1116] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, fortherapeutic purposes, for example, to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of wound healing,and to stimulate hair follicle production and healing of dermal wounds.Polynucleotides or polypeptides, as well as agonists or antagonists ofthe invention, may be clinically useful in stimulating wound healingincluding surgical wounds, excisional wounds, deep wounds involvingdamage of the dermis and epidermis, eye tissue wounds, dental tissuewounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitusulcers, arterial ulcers, venous stasis ulcers, burns resulting from heatexposure or chemicals, and other abnormal wound healing conditions suchas uremia, malnutrition, vitamin deficiencies and complicationsassociated with systemic treatment with steroids, radiation therapy andantineoplastic drugs and antimetabolites. Polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused to promote dermal reestablishment subsequent to denmal loss.

[1117] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used to increase the adherence ofskin grafts to a wound bed and to stimulate re-epithelialization fromthe wound bed. The following are a non-exhaustive list of grafts thatpolynucleotides or polypeptides, agonists or antagonists of theinvention, could be used to increase adherence to a wound bed:autografts, artificial skin, allografts, autodermic graft, autoepidermicgrafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplasticgrafts, cutis graft, delayed graft, dermic graft, epidermic graft,fascia graft, full thickness graft, heterologous graft, xenograft,homologous graft, hyperplastic graft, lamellar graft, mesh graft,mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft,pedicle graft, penetrating graft, split skin graft, thick split graft.The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, can be used to promote skin strength and to improve theappearance of aged skin.

[1118] It is believed that the polynucleotides or polypeptides, and/oragonists or antagonists of the invention, will also produce changes inhepatocyte proliferation, and epithelial cell proliferation in the lung,breast, pancreas, stomach, small intestine, and large intestine. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could promote proliferation of epithelial cells such assebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, may promote proliferation of endothelialcells, keratinocytes, and basal keratinocytes.

[1119] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could also be used to reduce the sideeffects of gut toxicity that result from radiation, chemotherapytreatments or viral infections. The polynucleotides or polypeptides,and/or agonists or antagonists of the invention, may have acytoprotective effect on the small intestine mucosa. The polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, mayalso stimulate healing of mucositis (mouth ulcers) that result fromchemotherapy and viral infections.

[1120] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could further be used in full regenerationof skin in full and partial thickness skin defects, including burns,(i.e., repopulation of hair follicles, sweat glands, and sebaceousglands), treatment of other skin defects such as psoriasis. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to treat epidermolysis bullosa, a defect inadherence of the epidermis to the underlying dermis which results infrequent, open and painful blisters by accelerating reepithelializationof these lesions. The polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, could also be used to treat gastric anddoudenal ulcers and help heal by scar formation of the mucosal liningand regeneration of glandular mucosa and duodenal mucosal lining morerapidly. Inflammatory bowel diseases, such as Crohn's disease andulcerative colitis, are diseases which result in destruction of themucosal surface of the small or large intestine, respectively. Thus, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to promote the resurfacing of the mucosalsurface to aid more rapid healing and to prevent progression ofinflammatory bowel disease. Treatment with the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, isexpected to have a significant effect on the production of mucusthroughout the gastrointestinal tract and could be used to protect theintestinal mucosa from injurious substances that are ingested orfollowing surgery. The polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, could be used to treat diseasesassociate with the under expression of the polynucleotides of theinvention.

[1121] Moreover, the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used to prevent and heal damageto the lungs due to various pathological states. A growth factor such asthe polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, which could stimulate proliferation and differentiationand promote the repair of alveoli and brochiolar epithelium to preventor treat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of alveoli, and inhalation injuries,i.e., resulting from smoke inhalation and burns, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treatedusing the polynucleotides or polypeptides, and/or agonists orantagonists of the invention. Also, the polynucleotides or polypeptides,and/or agonists or antagonists of the invention, could be used tostimulate the proliferation of and differentiation of type IIpneumocytes, which may help treat or prevent disease such as hyalinemembrane diseases, such as infant respiratory distress syndrome andbronchopulmonary dysplasia, in premature infants.

[1122] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could stimulate the proliferation anddifferentiation of hepatocytes and, thus, could be used to alleviate ortreat liver diseases and pathologies such as fulminant liver failurecaused by cirrhosis, liver damage caused by viral hepatitis and toxicsubstances (i.e., acetaminophen, carbon tetraholoride and otherhepatotoxins known in the art).

[1123] In addition, the polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, could be used treat or prevent theonset of diabetes mellitus. In patients with newly diagnosed Types I andII diabetes, where some islet cell function remains, the polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, couldbe used to maintain the islet function so as to alleviate, delay orprevent permanent manifestation of the disease. Also, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used as an auxiliary in islet cell transplantationto improve or promote islet cell function.

[1124] Neurological Diseases

[1125] Nervous system disorders, which can be treated with thecompositions of the invention (e.g., polypeptides, polynucleotides,and/or agonists or antagonists), include, but are not limited to,nervous system injuries, and diseases or disorders which result ineither a disconnection of axons, a diminution or degeneration ofneurons, or demyelination. Nervous system lesions which may be treatedin a patient (including human and non-human mammalian patients)according to the invention, include but are not limited to, thefollowing lesions of either the central (including spinal cord, brain)or peripheral nervous systems: (1) ischemic lesions, in which a lack ofoxygen in a portion of the nervous system results in neuronal injury ordeath, including cerebral infarction or ischemia, or spinal cordinfarction or ischemia; (2) traumatic lesions, including lesions causedby physical injury or associated with surgery, for example, lesionswhich sever a portion of the nervous system, or compression injuries;(3) malignant lesions, in which a portion of the nervous system isdestroyed or injured by malignant tissue which is either a nervoussystem associated malignancy or a malignancy derived from non-nervoussystem tissue; (4) infectious lesions, in which a portion of the nervoussystem is destroyed or injured as a result of infection, for example, byan abscess or associated with infection by human immunodeficiency virus,herpes zoster, or herpes simplex virus or with Lyme disease,tuberculosis, syphilis; (5) degenerative lesions, in which a portion ofthe nervous system is destroyed or injured as a result of a degenerativeprocess including but not limited to degeneration associated withParkinson's disease, Alzheimer's disease, Huntington's chorea, oramyotrophic lateral sclerosis (ALS); (6) lesions associated withnutritional diseases or disorders, in which a portion of the nervoussystem is destroyed or injured by a nutritional disorder or disorder ofmetabolism including but not limited to, vitamin B12 deficiency, folicacid deficiency, Wernicke disease, tobacco-alcohol amblyopia,Marchiafava-Bignami disease (primary degeneration of the corpuscallosum), and alcoholic cerebellar degeneration; (7) neurologicallesions associated with systemic diseases including, but not limited to,diabetes (diabetic neuropathy, Bell's palsy), systemic lupuserythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxicsubstances including alcohol, lead, or particular neurotoxins; and (9)demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

[1126] In a preferred embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to protect neuralcells from the damaging effects of cerebral hypoxia. According to thisembodiment, the compositions of the invention are used to treat orprevent neural cell injury associated with cerebral hypoxia. In oneaspect of this embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat or preventneural cell injury associated with cerebral ischemia. In another aspectof this embodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat or prevent neural cellinjury associated with cerebral infarction. In another aspect of thisembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat or prevent neural cellinjury associated with a stroke. In a further aspect of this embodiment,the polypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat or prevent neural cell injury associatedwith a heart attack.

[1127] The compositions of the invention which are useful for treatingor preventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture; (2) increased sprouting of neurons in culture or in vivo; (3)increased production of a neuron-associated molecule in culture or invivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, the method set forthin Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increasedsprouting of neurons may be detected by methods known in the art, suchas, for example, the methods set forth in Pestronk et al. (Exp. Neurol.70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 (1981));increased production of neuron-associated molecules may be measured bybioassay, enzymatic assay, antibody binding, Northern blot assay, etc.,using techniques known in the art and depending on the molecule to bemeasured; and motor neuron dysfunction may be measured by assessing thephysical manifestation of motor neuron disorder, e.g., weakness, motorneuron conduction velocity, or functional disability.

[1128] In specific embodiments, motor neuron disorders that may betreated according to the invention include, but are not limited to,disorders such as infarction, infection, exposure to toxin, trauma,surgical damage, degenerative disease or malignancy that may affectmotor neurons as well as other components of the nervous system, as wellas disorders that selectively affect neurons such as amyotrophic lateralsclerosis, and including, but not limited to, progressive spinalmuscular atrophy, progressive bulbar palsy, primary lateral sclerosis,infantile and juvenile muscular atrophy, progressive bulbar paralysis ofchildhood (Fazio-Londe syndrome), poliomyelitis and the post poliosyndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-ToothDisease).

[1129] Infectious Disease

[1130] A polypeptide or polynucleotide and/or agonist or antagonist ofthe present invention can be used to treat or detect infectious agents.For example, by increasing the immune response, particularly increasingthe proliferation and differentiation of B and/or T cells, infectiousdiseases may be treated. The immune response may be increased by eitherenhancing an existing immune response, or by initiating a new immuneresponse. Alternatively, polypeptide or polynucleotide and/or agonist orantagonist of the present invention may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

[1131] Viruses are one example of an infectious agent that can causedisease or symptoms that can be treated or detected by a polynucleotideor polypeptide and/or agonist or antagonist of the present invention.Examples of viruses, include, but are not limited to Examples ofviruses, include, but are not limited to the following DNA and RNAviruses and viral families: Arbovirus, Adenoviridae, Arenaviridae,Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae,Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae(Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex,Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, andparainfluenza), Papiloma virus, Papovaviridae, Parvoviridae,Picomaviridae, Poxyiridae (such as Smallpox or Vaccinia), Reoviridae(e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), andTogaviridae (e.g., Rubivirus). Viruses falling within these families cancause a variety of diseases or symptoms, including, but not limited to:arthritis, bronchiollitis, respiratory syncytial virus, encephalitis,eye infections (e.g., conjunctivitis, keratitis), chronic fatiguesyndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese Bencephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever,meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt'sLymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza,Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitteddiseases, skin diseases (e.g., Kaposi's, warts), and viremia.polynucleotides or polypeptides, or agonists or antagonists of theinvention, can be used to treat or detect any of these symptoms ordiseases. In specific embodiments, polynucleotides, polypeptides, oragonists or antagonists of the invention are used to treat: meningitis,Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additionalspecific embodiment polynucleotides, polypeptides, or agonists orantagonists of the invention are used to treat patients nonresponsive toone or more other commercially available hepatitis vaccines. In afurther specific embodiment polynucleotides, polypeptides, or agonistsor antagonists of the invention are used to treat AIDS.

[1132] Similarly, bacterial or fungal agents that can cause disease orsymptoms and that can be treated or detected by a polynucleotide orpolypeptide and/or agonist or antagonist of the present inventioninclude, but not limited to, include, but not limited to, the followingGram-Negative and Gram-positive bacteria and bacterial families andfungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium,Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g.,Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis,Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E.coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, andSalmonella paratyphi), Serratia, Yersinia), Erysipelothrix,Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g.,Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fungalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., meningitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Polynucleotides or polypeptides, agonists orantagonists of the invention, can be used to treat or detect any ofthese symptoms or diseases. In specific embodiments, Polynucleotides,polypeptides, agonists or antagonists of the invention are used totreat: tetanus, Diptheria, botulism, and/or meningitis type B.

[1133] Moreover, parasitic agents causing disease or symptoms that canbe treated or detected by a polynucleotide or polypeptide and/or agonistor antagonist of the present invention include, but not limited to, thefollowing families or class: Amebiasis, Babesiosis, Coccidiosis,Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis,Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), malaria, pregnancycomplications, and toxoplasmosis. polynucleotides or polypeptides, oragonists or antagonists of the invention, can be used to treat or detectany of these symptoms or diseases. In specific embodiments,polynucleotides, polypeptides, or agonists or antagonists of theinvention are used to treat malaria.

[1134] Preferably, treatment using a polypeptide or polynucleotideand/or agonist or antagonist of the present invention could either be byadministering an effective amount of a polypeptide to the patient, or byremoving cells from the patient, supplying the cells with apolynucleotide of the present invention, and returning the engineeredcells to the patient (ex vivo therapy). Moreover, the polypeptide orpolynucleotide of the present invention can be used as an antigen in avaccine to raise an immune response against infectious disease.

[1135] Regeneration

[1136] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention can be used to differentiate, proliferate, andattract cells, leading to the regeneration of tissues. (See, Science276:59-87 (1997).) The regeneration of tissues could be used to repair,replace, or protect tissue damaged by congenital defects, trauma(wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis,osteocarthritis, periodontal disease, liver failure), surgery, includingcosmetic plastic surgery, fibrosis, reperfusion injury, or systemiccytokine damage.

[1137] Tissues that could be regenerated using the present inventioninclude organs (e.g., pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac), vasculature(including vascular and lymphatics), nervous, hematopoietic, andskeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,regeneration occurs without or decreased scarring. Regeneration also mayinclude angiogenesis.

[1138] Moreover, a polynucleotide or polypeptide and/or agonist orantagonist of the present invention may increase regeneration of tissuesdifficult to heal. For example, increased tendon/ligament regenerationwould quicken recovery time after damage. A polynucleotide orpolypeptide and/or agonist or antagonist of the present invention couldalso be used prophylactically in an effort to avoid damage. Specificdiseases that could be treated include of tendinitis, carpal tunnelsyndrome, and other tendon or ligament defects. A further example oftissue regeneration of non-healing wounds includes pressure ulcers,ulcers associated with vascular insufficiency, surgical, and traumaticwounds.

[1139] Similarly, nerve and brain tissue could also be regenerated byusing a polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention to proliferate and differentiate nerve cells.Diseases that could be treated using this method include central andperipheral nervous system diseases, neuropathies, or mechanical andtraumatic disorders (e.g., spinal cord disorders, head trauma,cerebrovascular disease, and stoke). Specifically, diseases associatedwith peripheral nerve injuries, peripheral neuropathy (e.g., resultingfrom chemotherapy or other medical therapies), localized neuropathies,and central nervous system diseases (e.g., Alzheimer's disease,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome), could all be treated using thepolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention.

[1140] Chemotaxis

[1141] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may have chemotaxis activity. A chemotaxicmolecule attracts or mobilizes cells (e.g., monocytes, fibroblasts,neutrophils, T-cells, mast cells, eosinophils, epithelial and/orendothelial cells) to a particular site in the body, such asinflammation, infection, or site of hyperproliferation. The mobilizedcells can then fight off and/or heal the particular trauma orabnormality.

[1142] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase chemotaxic activity of particularcells. These chemotactic molecules can then be used to treatinflammation, infection, hyperproliferative disorders, or any immunesystem disorder by increasing the number of cells targeted to aparticular location in the body. For example, chemotaxic molecules canbe used to treat wounds and other trauma to tissues by attracting immunecells to the injured location. Chemotactic molecules of the presentinvention can also attract fibroblasts, which can be used to treatwounds.

[1143] It is also contemplated that a polynucleotide or polypeptideand/or agonist or antagonist of the present invention may inhibitchemotactic activity. These molecules could also be used to treatdisorders. Thus, a polynucleotide or polypeptide and/or agonist orantagonist of the present invention could be used as an inhibitor ofchemotaxis.

[1144] Binding Activity

[1145] A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors), orsmall molecules.

[1146] Preferably, the molecule is closely related to the natural ligandof the polypeptide, e.g., a fragment of the ligand, or a naturalsubstrate, a ligand, a structural or functional mimetic. (See, Coliganet al., Current Protocols in Immunology 1(2):Chapter 5 (1991).)Similarly, the molecule can be closely related to the natural receptorto which the polypeptide binds, or at least, a fragment of the receptorcapable of being bound by the polypeptide (e.g., active site). In eithercase, the molecule can be rationally designed using known techniques.

[1147] Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofeither the polypeptide or the molecule.

[1148] The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

[1149] Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

[1150] Preferably, an ELISA assay can measure polypeptide level oractivity in a sample (e.g., biological sample) using a monoclonal orpolyclonal antibody. The antibody can measure polypeptide level oractivity by either binding, directly or indirectly, to the polypeptideor by competing with the polypeptide for a substrate.

[1151] Additionally, the receptor to which a polypeptide of theinvention binds can be identified by numerous methods known to those ofskill in the art, for example, ligand panning and FACS sorting (Coligan,et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Forexample, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the polypeptides, for example, NIH3T3cells which are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labeled. The polypeptides can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase.

[1152] Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

[1153] As an alternative approach for receptor identification, thelabeled polypeptides can be photoaffinity linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE analysis and exposed to X-ray film. Thelabeled complex containing the receptors of the polypeptides can beexcised, resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

[1154] Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of polypeptidesof the invention thereby effectively generating agonists and antagonistsof polypeptides of the invention. See generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten,P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S.Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol.Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques24(2):308-13 (1998) (each of these patents and publications are herebyincorporated by reference). In one embodiment, alteration ofpolynucleotides and corresponding polypeptides of the invention may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments into a desired polynucleotide sequence of theinvention molecule by homologous, or site-specific, recombination. Inanother embodiment, polynucleotides and corresponding polypeptides ofthe invention may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of the polypeptides of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. In preferred embodiments, the heterologous molecules arefamily members. In further preferred embodiments, the heterologousmolecule is a growth factor such as, for example, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-I), transforminggrowth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblastgrowth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2,BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic (dpp),60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

[1155] Other preferred fragments are biologically active fragments ofthe polypeptides of the invention. Biologically active fragments arethose exhibiting activity similar, but not necessarily identical, to anactivity of the polypeptide. The biological activity of the fragmentsmay include an improved desired activity, or a decreased undesirableactivity.

[1156] Additionally, this invention provides a method of screeningcompounds to identify those which modulate the action of the polypeptideof the present invention. An example of such an assay comprisescombining a mammalian fibroblast cell, a the polypeptide of the presentinvention, the compound to be screened and 3[H] thymidine under cellculture conditions where the fibroblast cell would normally proliferate.A control assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of 3[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of 3[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

[1157] In another method, a mammalian cell or membrane preparationexpressing a receptor for a polypeptide of the present invention isincubated with a labeled polypeptide of the present invention in thepresence of the compound. The ability of the compound to enhance orblock this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction of acompound to be screened and the receptor is measured and the ability ofthe compound to bind to the receptor and elicit a second messengerresponse is measured to determine if the compound is a potential agonistor antagonist. Such second messenger systems include but are not limitedto, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

[1158] All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting thepolypeptide/molecule. Moreover, the assays can discover agents which mayinhibit or enhance the production of the polypeptides of the inventionfrom suitably manipulated cells or tissues. Therefore, the inventionincludes a method of identifying compounds which bind to thepolypeptides of the invention comprising the steps of: (a) incubating acandidate binding compound with the polypeptide; and (b) determining ifbinding has occurred. Moreover, the invention includes a method ofidentifying agonists/antagonists comprising the steps of: (a) incubatinga candidate compound with the polypeptide, (b) assaying a biologicalactivity, and (b) determining if a biological activity of thepolypeptide has been altered.

[1159] Also, one could identify molecules bind a polypeptide of theinvention experimentally by using the beta-pleated sheet regionscontained in the polypeptide sequence of the protein. Accordingly,specific embodiments of the invention are directed to polynucleotidesencoding polypeptides which comprise, or alternatively consist of, theamino acid sequence of each beta pleated sheet regions in a disclosedpolypeptide sequence. Additional embodiments of the invention aredirected to polynucleotides encoding polypeptides which comprise, oralternatively consist of, any combination or all of contained in thepolypeptide sequences of the invention. Additional preferred embodimentsof the invention are directed to polypeptides which comprise, oralternatively consist of, the amino acid sequence of each of the betapleated sheet regions in one of the polypeptide sequences of theinvention. Additional embodiments of the invention are directed topolypeptides which comprise, or alternatively consist of, anycombination or all of the beta pleated sheet regions in one of thepolypeptide sequences of the invention.

[1160] Targeted Delivery

[1161] In another embodiment, the invention provides a method ofdelivering compositions to targeted cells expressing a receptor for apolypeptide of the invention, or cells expressing a cell bound form of apolypeptide of the invention.

[1162] As discussed herein, polypeptides or antibodies of the inventionmay be associated with heterologous polypeptides, heterologous nucleicacids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/orcovalent interactions. In one embodiment, the invention provides amethod for the specific delivery of compositions of the invention tocells by administering polypeptides of the invention (includingantibodies) that are associated with heterologous polypeptides ornucleic acids. In one example, the invention provides a method fordelivering a therapeutic protein into the targeted cell. In anotherexample, the invention provides a method for delivering a singlestranded nucleic acid (e.g., antisense or ribozymes) or double strandednucleic acid (e.g., DNA that can integrate into the cell's genome orreplicate episomally and that can be transcribed) into the targetedcell.

[1163] In another embodiment, the invention provides a method for thespecific destruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

[1164] By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

[1165] Drug Screening

[1166] Further contemplated is the use of the polypeptides of thepresent invention, or the polynucleotides encoding these polypeptides,to screen for molecules which modify the activities of the polypeptidesof the present invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

[1167] This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

[1168] Thus, the present invention provides methods of screening fordrugs or any other agents which affect activities mediated by thepolypeptides of the present invention. These methods comprise contactingsuch an agent with a polypeptide of the present invention or a fragmentthereof and assaying for the presence of a complex between the agent andthe polypeptide or a fragment thereof, by methods well known in the art.In such a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

[1169] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to thepolypeptides of the present invention, and is described in great detailin European Patent Application 84/03564, published on Sep. 13, 1984,which is incorporated herein by reference herein. Briefly stated, largenumbers of different small peptide test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The peptidetest compounds are reacted with polypeptides of the present inventionand washed. Bound polypeptides are then detected by methods well knownin the art. Purified polypeptides are coated directly onto plates foruse in the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

[1170] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of bindingpolypeptides of the present invention specifically compete with a testcompound for binding to the polypeptides or fragments thereof. In thismanner, the antibodies are used to detect the presence of any peptidewhich shares one or more antigenic epitopes with a polypeptide of theinvention.

[1171] Antisense And Ribozyme (Antagonists)

[1172] In specific embodiments, antagonists according to the presentinvention are nucleic acids corresonding to the sequences contained inSEQ ID NO:X, or the complementary strand thereof, and/or to nucleotidesequences contained a deposited clone. In one embodiment, antisensesequence is generated internally by the organism, in another embodiment,the antisense sequence is separately administered (see, for example,O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as AnitsenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Antisense technology can be used to control gene expression throughantisense DNA or RNA, or through triple-helix formation. Antisensetechniques are discussed for example, in Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research,6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan etal., Science, 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

[1173] For example, the use of c-myc and c-myb antisense RNA constructsto inhibit the growth of the non-lymphocytic leukemia cell line HL-60and other cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcoR1 site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5,10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligatedto the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

[1174] For example, the 5′ coding portion of a polynucleotide thatencodes the mature polypeptide of the present invention may be used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

[1175] In one embodiment, the antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the antisense nucleic acid of the invention.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding a polypeptide of the invention, orfragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinsteret al., Nature, 296:39-42 (1982)), etc.

[1176] The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a gene ofinterest. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded antisense nucleic acids of the invention, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acidGenerally, the larger the hybridizing nucleic acid, the more basemismatches with a RNA sequence of the invention it may contain and stillform a stable duplex (or triplex as the case may be). One skilled in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

[1177] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature,372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′-non-translated, non-coding regions of a polynucleotidesequence of the invention could be used in an antisense approach toinhibit translation of endogenous mRNA. Oligonucleotides complementaryto the 5′ untranslated region of the mRNA should include the complementof the AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the invention. Whether designed to hybridize tothe 5′-, 3′- or coding region of mRNA, antisense nucleic acids should beat least six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides or at least 50 nucleotides.

[1178] The polynucleotides of the invention can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, etc. Theoligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al.,Proc. Natl. Acad. Sci., 84:648-652 (1987); PCT Publication NO:WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see,e.g., PCT Publication NO: WO89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents. (See, e.g., Krol et al.,BioTechniques, 6:958-976 (1988)) or intercalating agents. (See, e.g.,Zon, Pharm. Res., 5:539-549 (1988)). To this end, the oligonucleotidemay be conjugated to another molecule, e.g., a peptide, hybridizationtriggered cross-linking agent, transport agent, hybridization-triggeredcleavage agent, etc.

[1179] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including, but not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[1180] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[1181] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[1182] In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a2-O-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

[1183] Polynucleotides of the invention may be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci.U.S.A., 85:7448-7451 (1988)), etc.

[1184] While antisense nucleotides complementary to the coding regionsequence of the invention could be used, those complementary to thetranscribed untranslated region are most preferred.

[1185] Potential antagonists according to the invention also includecatalytic RNA, or a ribozyme (See, e.g., PCT International PublicationWO 90/11364, published Oct. 4, 1990; Sarver et al, Science,247:1222-1225 (1990). While ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy mRNAs corresponding to thepolynucleotides of the invention, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within eachnucleotide sequence disclosed in the sequence listing. Preferably, theribozyme is engineered so that the cleavage recognition site is locatednear the 5′ end of the mRNA corresponding to the polynucleotides of theinvention; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

[1186] As in the antisense approach, the ribozymes of the invention canbe composed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express thepolynucleotides of the invention in vivo. DNA constructs encoding theribozyme may be introduced into the cell in the same manner as describedabove for the introduction of antisense encoding DNA. A preferred methodof delivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive promoter, such as, for example, polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous messages andinhibit translation. Since ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

[1187] Antagonist/agonist compounds may be employed to inhibit the cellgrowth and proliferation effects of the polypeptides of the presentinvention on neoplastic cells and tissues, i.e. stimulation ofangiogenesis of tumors, and, therefore, retard or prevent abnormalcellular growth and proliferation, for example, in tumor formation orgrowth.

[1188] The antagonist/agonist may also be employed to preventhyper-vascular diseases, and prevent the proliferation of epitheliallens cells after extracapsular cataract surgery. Prevention of themitogenic activity of the polypeptides of the present invention may alsobe desirous in cases such as restenosis after balloon angioplasty.

[1189] The antagonist/agonist may also be employed to prevent the growthof scar tissue during wound healing.

[1190] The antagonist/agonist may also be employed to treat the diseasesdescribed herein.

[1191] Thus, the invention provides a method of treating disorders ordiseases, including but not limited to the disorders or diseases listedthroughout this application, associated with overexpression of apolynucleotide of the present invention by administering to a patient(a) an antisense molecule directed to the polynucleotide of the presentinvention, and/or (b) a ribozyme directed to the polynucleotide of thepresent invention

[1192] Other Activities

[1193] The polypeptide of the present invention, as a result of theability to stimulate vascular endothelial cell growth, may be employedin treatment for stimulating re-vascularization of ischemic tissues dueto various disease conditions such as thrombosis, arteriosclerosis, andother cardiovascular conditions. These polypeptide may also be employedto stimulate angiogenesis and limb regeneration, as discussed above.

[1194] The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

[1195] The polypeptide of the present invention may also be employedstimulate neuronal growth and to treat and prevent neuronal damage whichoccurs in certain neuronal disorders or neuro-degenerative conditionssuch as Alzheimer's disease, Parkinson's disease, and AIDS-relatedcomplex. The polypeptide of the invention may have the ability tostimulate chondrocyte growth, therefore, they may be employed to enhancebone and periodontal regeneration and aid in tissue transplants or bonegrafts.

[1196] The polypeptide of the present invention may be also be employedto prevent skin aging due to sunburn by stimulating keratinocyte growth.

[1197] The polypeptide of the invention may also be employed forpreventing hair loss, since FGF family members activate hair-formingcells and promotes melanocyte growth. Along the same lines, thepolypeptides of the present invention may be employed to stimulategrowth and differentiation of hematopoietic cells and bone marrow cellswhen used in combination with other cytokines.

[1198] The polypeptide of the invention may also be employed to maintainorgans before transplantation or for supporting cell culture of primarytissues.

[1199] The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

[1200] The polypeptide or polynucleotides and/or agonist or antagonistsof the present invention may also increase or decrease thedifferentiation or proliferation of embryonic stem cells, besides, asdiscussed above, hematopoietic lineage.

[1201] The polypeptide or polynucleotides and/or agonist or antagonistsof the present invention may also be used to modulate mammaliancharacteristics, such as body height, weight, hair color, eye color,skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,cosmetic surgery). Similarly, polypeptides or polynucleotides and/oragonist or antagonists of the present invention may be used to modulatemammalian metabolism affecting catabolism, anabolism, processing,utilization, and storage of energy.

[1202] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may be used to change a mammal's mental state orphysical state by influencing biorhythms, circadian rhythms, depression(including depressive disorders), tendency for violence, tolerance forpain, reproductive capabilities (preferably by Activin or Inhibin-likeactivity), hormonal or endocrine levels, appetite, libido, memory,stress, or other cognitive qualities.

[1203] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used as a food additive orpreservative, such as to increase or decrease storage capabilities, fatcontent, lipid, protein, carbohydrate, vitamins, minerals, cofactors orother nutritional components.

[1204] Other Preferred Embodiments

[1205] Other preferred embodiments of the claimed invention include anisolated nucleic acid molecule comprising a nucleotide sequence which isat least 95% identical to a sequence of at least about 50 contiguousnucleotides in the nucleotide sequence of SEQ ID NO:X wherein X is anyinteger as defined in Table 1.

[1206] Also preferred is a nucleic acid molecule wherein said sequenceof contiguous nucleotides is included in the nucleotide sequence of SEQID NO:X in the range of positions beginning with the nucleotide at aboutthe position of the 5′ Nucleotide of the Clone Sequence and ending withthe nucleotide at about the position of the 3′ Nucleotide of the CloneSequence as defined for SEQ ID NO:X in Table 1.

[1207] Also preferred is a nucleic acid molecule wherein said sequenceof contiguous nucleotides is included in the nucleotide sequence of SEQID NO:X in the range of positions beginning with the nucleotide at aboutthe position of the 5′ Nucleotide of the Start Codon and ending with thenucleotide at about the position of the 3′ Nucleotide of the CloneSequence as defined for SEQ ID NO:X in Table 1.

[1208] Similarly preferred is a nucleic acid molecule wherein saidsequence of contiguous nucleotides is included in the nucleotidesequence of SEQ ID NO:X in the range of positions beginning with thenucleotide at about the position of the 5′ Nucleotide of the First AminoAcid of the Signal Peptide and ending with the nucleotide at about theposition of the 3′ Nucleotide of the Clone Sequence as defined for SEQID NO:X in Table 1.

[1209] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 150 contiguous nucleotides in the nucleotide sequence of SEQID NO:X.

[1210] Further preferred is an isolated nucleic acid molecule comprisinga nucleotide sequence which is at least 95% identical to a sequence ofat least about 500 contiguous nucleotides in the nucleotide sequence ofSEQ ID NO:X.

[1211] A further preferred embodiment is a nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thenucleotide sequence of SEQ ID NO:X beginning with the nucleotide atabout the position of the 5′ Nucleotide of the First Amino Acid of theSignal Peptide and ending with the nucleotide at about the position ofthe 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X inTable 1.

[1212] A further preferred embodiment is an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to the complete nucleotide sequence of SEQ ID NO:X.

[1213] Also preferred is an isolated nucleic acid molecule whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule, wherein said nucleic acid molecule which hybridizes does nothybridize under stringent hybridization conditions to a nucleic acidmolecule having a nucleotide sequence consisting of only A residues orof only T residues.

[1214] Also preferred is a composition of matter comprising a DNAmolecule which comprises a human cDNA clone identified by a cDNA CloneIdentifier in Table 1, which DNA molecule is contained in the materialdeposited with the American Type Culture Collection and given the ATCCDeposit Number shown in Table 1 for said cDNA Clone Identifier.

[1215] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in the nucleotide sequence of a humancDNA clone identified by a cDNA Clone Identifier in Table 1, which DNAmolecule is contained in the deposit given the ATCC Deposit Number shownin Table 1.

[1216] Also preferred is an isolated nucleic acid molecule, wherein saidsequence of at least 50 contiguous nucleotides is included in thenucleotide sequence of the complete open reading frame sequence encodedby said human cDNA clone.

[1217] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to sequence of atleast 150 contiguous nucleotides in the nucleotide sequence encoded bysaid human cDNA clone.

[1218] A further preferred embodiment is an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to sequence of at least 500 contiguous nucleotides in thenucleotide sequence encoded by said human cDNA clone.

[1219] A further preferred embodiment is an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to the complete nucleotide sequence encoded by said human cDNAclone.

[1220] A further preferred embodiment is a method for detecting in abiological sample a nucleic acid molecule comprising a nucleotidesequence which is at least 95% identical to a sequence of at least 50contiguous nucleotides in a sequence selected from the group consistingof: a nucleotide sequence of SEQ ID NO:X wherein X is any integer asdefined in Table 1; and a nucleotide sequence encoded by a human cDNAclone identified by a cDNA Clone Identifier in Table 1 and contained inthe deposit with the ATCC Deposit Number shown for said cDNA clone inTable 1; which method comprises a step of comparing a nucleotidesequence of at least one nucleic acid molecule in said sample with asequence selected from said group and determining whether the sequenceof said nucleic acid molecule in said sample is at least 95% identicalto said selected sequence.

[1221] Also preferred is the above method wherein said step of comparingsequences comprises determining the extent of nucleic acid hybridizationbetween nucleic acid molecules in said sample and a nucleic acidmolecule comprising said sequence selected from said group. Similarly,also preferred is the above method wherein said step of comparingsequences is performed by comparing the nucleotide sequence determinedfrom a nucleic acid molecule in said sample with said sequence selectedfrom said group. The nucleic acid molecules can comprise DNA moleculesor RNA molecules.

[1222] A further preferred embodiment is a method for identifying thespecies, tissue or cell type of a biological sample which methodcomprises a step of detecting nucleic acid molecules in said sample, ifany, comprising a nucleotide sequence that is at least 95% identical toa sequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:Xwherein X is any integer as defined in Table 1; and a nucleotidesequence encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1.

[1223] The method for identifying the species, tissue or cell type of abiological sample can comprise a step of detecting nucleic acidmolecules comprising a nucleotide sequence in a panel of at least twonucleotide sequences, wherein at least one sequence in said panel is atleast 95% identical to a sequence of at least 50 contiguous nucleotidesin a sequence selected from said group.

[1224] Also preferred is a method for diagnosing in a subject apathological condition associated with abnormal structure or expressionof a gene encoding a secreted protein identified in Table 1, whichmethod comprises a step of detecting in a biological sample obtainedfrom said subject nucleic acid molecules, if any, comprising anucleotide sequence that is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from the groupconsisting of: a nucleotide sequence of SEQ ID NO:X wherein X is anyinteger as defined in Table 1; and a nucleotide sequence encoded by ahuman cDNA clone identified by a cDNA Clone Identifier in Table 1 andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table 1.

[1225] The method for diagnosing a pathological condition can comprise astep of detecting nucleic acid molecules comprising a nucleotidesequence in a panel of at least two nucleotide sequences, wherein atleast one sequence in said panel is at least 95% identical to a sequenceof at least 50 contiguous nucleotides in a sequence selected from saidgroup.

[1226] Also preferred is a composition of matter comprising isolatednucleic acid molecules wherein the nucleotide sequences of said nucleicacid molecules comprise a panel of at least two nucleotide sequences,wherein at least one sequence in said panel is at least 95% identical toa sequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:Xwherein X is any integer as defined in Table 1; and a nucleotidesequence encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1. The nucleic acid moleculescan comprise DNA molecules or RNA molecules.

[1227] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the amino acid sequence of SEQ ID NO:Y whereinY is any integer as defined in Table 1.

[1228] Also preferred is a polypeptide, wherein said sequence ofcontiguous amino acids is included in the amino acid sequence of SEQ IDNO:Y in the range of positions beginning with the residue at about theposition of the First Amino Acid of the Secreted Portion and ending withthe residue at about the Last Amino Acid of the Open Reading Frame asset forth for SEQ ID NO:Y in Table 1.

[1229] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

[1230] Further preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

[1231] Further preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to the complete amino acid sequenceof SEQ ID NO:Y.

[1232] Further preferred is an isolated polypeptide comprising an aminoacid sequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the complete amino acid sequence of a secretedprotein encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1.

[1233] Also preferred is a polypeptide wherein said sequence ofcontiguous amino acids is included in the amino acid sequence of asecreted portion of the secreted protein encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table 1 and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in Table1.

[1234] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of the secretedportion of the protein encoded by a human cDNA clone identified by acDNA Clone Identifier in Table 1 and contained in the deposit with theATCC Deposit Number shown for said cDNA clone in Table 1.

[1235] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of the secretedportion of the protein encoded by a human cDNA clone identified by acDNA Clone Identifier in Table 1 and contained in the deposit with theATCC Deposit Number shown for said cDNA clone in Table 1.

[1236] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to the amino acid sequence of thesecreted portion of the protein encoded by a human cDNA clone identifiedby a cDNA Clone Identifier in Table 1 and contained in the deposit withthe ATCC Deposit Number shown for said cDNA clone in Table 1.

[1237] Further preferred is an isolated antibody which bindsspecifically to a polypeptide comprising an amino acid sequence that isat least 90% identical to a sequence of at least 10 contiguous aminoacids in a sequence selected from the group consisting of: an amino acidsequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1;and a complete amino acid sequence of a protein encoded by a human cDNAclone identified by a cDNA Clone Identifier in Table 1 and contained inthe deposit with the ATCC Deposit Number shown for said cDNA clone inTable 1.

[1238] Further preferred is a method for detecting in a biologicalsample a polypeptide comprising an amino acid sequence which is at least90% identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and acomplete amino acid sequence of a protein encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table 1 and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in Table1; which method comprises a step of comparing an amino acid sequence ofat least one polypeptide molecule in said sample with a sequenceselected from said group and determining whether the sequence of saidpolypeptide molecule in said sample is at least 90% identical to saidsequence of at least 10 contiguous amino acids.

[1239] Also preferred is the above method wherein said step of comparingan amino acid sequence of at least one polypeptide molecule in saidsample with a sequence selected from said group comprises determiningthe extent of specific binding of polypeptides in said sample to anantibody which binds specifically to a polypeptide comprising an aminoacid sequence that is at least 90% identical to a sequence of at least10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table 1; and a complete amino acid sequence of aprotein encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1.

[1240] Also preferred is the above method wherein said step of comparingsequences is performed by comparing the amino acid sequence determinedfrom a polypeptide molecule in said sample with said sequence selectedfrom said group.

[1241] Also preferred is a method for identifying the species, tissue orcell type of a biological sample which method comprises a step ofdetecting polypeptide molecules in said sample, if any, comprising anamino acid sequence that is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table 1; and a complete amino acid sequence of asecreted protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table 1 and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1.

[1242] Also preferred is the above method for identifying the species,tissue or cell type of a biological sample, which method comprises astep of detecting polypeptide molecules comprising an amino acidsequence in a panel of at least two amino acid sequences, wherein atleast one sequence in said panel is at least 90% identical to a sequenceof at least 10 contiguous amino acids in a sequence selected from theabove group.

[1243] Also preferred is a method for diagnosing in a subject apathological condition associated with abnormal structure or expressionof a gene encoding a secreted protein identified in Table 1, whichmethod comprises a step of detecting in a biological sample obtainedfrom said subject polypeptide molecules comprising an amino acidsequence in a panel of at least two amino acid sequences, wherein atleast one sequence in said panel is at least 90% identical to a sequenceof at least 10 contiguous amino acids in a sequence selected from thegroup consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y isany integer as defined in Table 1; and a complete amino acid sequence ofa secreted protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table 1 and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1.

[1244] In any of these methods, the step of detecting said polypeptidemolecules includes using an antibody.

[1245] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a nucleotidesequence encoding a polypeptide wherein said polypeptide comprises anamino acid sequence that is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table 1; and a complete amino acid sequence of asecreted protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table 1 and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1.

[1246] Also preferred is an isolated nucleic acid molecule, wherein saidnucleotide sequence encoding a polypeptide has been optimized forexpression of said polypeptide in a prokaryotic host.

[1247] Also preferred is an isolated nucleic acid molecule, wherein saidpolypeptide comprises an amino acid sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table 1; and a complete amino acid sequence of asecreted protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table 1 and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1.

[1248] Further preferred is a method of making a recombinant vectorcomprising inserting any of the above isolated nucleic acid moleculeinto a vector. Also preferred is the recombinant vector produced by thismethod. Also preferred is a method of making a recombinant host cellcomprising introducing the vector into a host cell, as well as therecombinant host cell produced by this method.

[1249] Also preferred is a method of making an isolated polypeptidecomprising culturing this recombinant host cell under conditions suchthat said polypeptide is expressed and recovering said polypeptide. Alsopreferred is this method of making an isolated polypeptide, wherein saidrecombinant host cell is a eukaryotic cell and said polypeptide is asecreted portion of a human secreted protein comprising an amino acidsequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:Y beginning with the residue at the position of the FirstAmino Acid of the Secreted Portion of SEQ ID NO:Y wherein Y is aninteger set forth in Table 1 and said position of the First Amino Acidof the Secreted Portion of SEQ ID NO:Y is defined in Table 1; and anamino acid sequence of a secreted portion of a protein encoded by ahuman cDNA clone identified by a cDNA Clone Identifier in Table 1 andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table 1. The isolated polypeptide produced by this methodis also preferred.

[1250] Also preferred is a method of treatment of an individual in needof an increased level of a secreted protein activity, which methodcomprises administering to such an individual a pharmaceuticalcomposition comprising an amount of an isolated polypeptide,polynucleotide, or antibody of the claimed invention effective toincrease the level of said protein activity in said individual.

[1251] The above-recited applications have uses in a wide variety ofhosts. Such hosts include, but are not limited to, human, murine,rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig,micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, andhuman. In specific embodiments, the host is a mouse, rabbit, goat,guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferredembodiments, the host is a mammal. In most preferred embodiments, thehost is a human.

[1252] In specific embodiments of the invention, for each “Contig ID”listed in the fourth column of Table 2, preferably excluded are one ormore polynucleotides comprising, or alternatively consisting of, anucleotide sequence referenced in the fifth column of Table 2 anddescribed by the general formula of a-b, whereas a and b are uniquelydetermined for the corresponding SEQ ID NO:X referred to in column 3 ofTable 2. Further specific embodiments are directed to polynucleotidesequences excluding one, two, three, four, or more of the specificpolynucleotide sequences referred to in the fifth column of Table 2. Inno way is this listing meant to encompass all of the sequences which maybe excluded by the general formula, it is just a representative example.All references available through these accessions are herebyincorporated by reference in their entirety. TABLE 2 NT SEQ ID cDNAClone NO: Gene No. ID X Contig ID Public Accession Numbers  1 HWBBP10 11 866121 None  1 HWBBP10 105 689013 None  2 HWBDO80  12 689154 None  3HWHGU54  13 695695 AA458648  4 HYACI76  14 695661 N49211, W93807,AA251779  5 HBHMA23  15 848016 D45555  5 HBHMA23 106 699815 None  6HCE3G20  16 699650 R10120, R19673, R20275, R38059, R38150, R43614,R44228, R44228, R43614, R68819, R68927, H06874, H06917, W86919, W96476,W96509, AA160664  7 HCEJP80  17 701975 None  8 HCUDD24  18 696674 None 9 HDPTD15  19 692917 None 10 HDPWU34  20 630354 T64144, T64272, R09211,R09320, N50984, AA242853, AA252172 10 HDPWU34 107 701979 None 11 HEOOV79 21 698965 R82970, H49748, H67534, H67535, H94998, H67535, N58918,N66579, N77856, N98839, W23717, W35317, W61140, W61178, AA037069,AA133772, AA133771, AA419253 12 HFKET93  22 690913 AA053057, AA131111,AA132859 13 HFTDL56  23 695976 None 14 HFXJX44  24 701988 None 15HKACU58  25 866118 None 15 HKACU58 108 698205 T97811, R17346, R21687,R37180, R42694, R46579, R42694, R46579, H11388, H11412, H41887, H46783,H50928, H60993, H68219, H68219, N25268, N33909, W94891, W92055,AA010304, AA010303, AA027849, AA027864, AA046120, AA046229, AA135112,AA135267, AA233866, AA256904, AA255480, AA258670, AA418852, AA418968,AA425788, AA424927 16 HKFBC53  26 701893 N33565, N50101 16 HLDBQ19 109513190 R36266, R88936, R90773, N72164, AA114066, AA115762, AA253139 16HLDBQ19 110 383426 None 17 HLTHR66  27 699812 R68887, R77685, R77684,H19020, H19313, AA026012, AA026000, AA084602 18 HLYBA69  28 701999 None19 HNTMX29  29 866388 None 19 HNTMX29 111 702008 None 20 HNTNC20  30700627 AA131240, AA131359 21 HNTNI01  31 699848 R13985 22 HOHCK70  32702010 None 23 HSMBE69  33 699878 T40515, T40518, T40517, T40525,T40530, R28533, R28674, R35242, R35243, R47799, R50432, R80602, R80603,R81797, R81901, AA043782, AA213657, AA261857, AA261858 24 HT4FW61  34699806 None 25 HYABK95  35 696650 T48366, R14541, R93472, H77708, H8565626 HYACE88  36 696962 T53019, T53020, T57689, T57729, T59933, T60002,R43268, R43268, R70761, R70810, R73004, H08232, R92213, R93957, R97093,H85350, H85764, H98074, N35624, N36006, N54914, N67623, N69761, N93791,AA064831, AA151643, AA151816, AA152197, AA157131 27 HOABR60  37 861308R82758, W37997, W37998, AA195463, AA252544, AA625460, AA844541, AA85354027 HOABR60 112 665765 AA252544 28 HAGCT73  38 638549 AA035336, AA044767,AA136244 29 HAPOM45  39 637867 R63388, R63443, R76013 30 HCEJQ69  40609999 None 31 HAGFI62  41 704425 T88930, R39072, R52911, R56893,R67277, H44134, H49659, AA009946 32 HAGGS43  42 704057 None 33 HBJHP03 43 704059 None 34 HCHPF68  44 704066 None 35 HDPJF37  45 704487 R74203,R74295 36 HSDEZ20  46 704101 None 37 HTEKU58  47 694601 None 38 HLTBL58 48 703759 None 39 HPWDJ42  49 722246 None 39 HPWDJ42 113 709662 None 39HPWDJ42 114 692213 None 40 HRACD15  50 871221 H47888, H89133, N54250,N81046, W07856, W60024, W60104, W96176, N90686, AA036807, AA037397,AA135546, AA236044, AA261854, AA261853, AA262692, AA622914, AA806404,AA830894, AA938381, AA994223, AA204918, AA643222, AA077110, AA077296,AA077415, AA078651, AA283856, AA283855, AA401964, AA402082, AA455506,AA455507, AA496293, AA496322, AA628809, AA778244, AA928793, AI075157,T10368, T10369, AA699788, AI262893, AI287716, AI202252, AI214676,AI452870, AI498229, AI189756, AI193362, AI214884, AI217271 40 HRACD15115 706332 H47888, H89133, N54250, N81046, W07856, W60024, W60104,W96176, N90686, AA036807, AA037397, AA135546, AA236044, AA261854,AA261853, AA262692 41 HSIAC80  51 709682 None 42 HAGFD18  52 720478R11835, R18441, R36954, R52768, R54314, H08720, H15365, H18641, N22295,N81196 43 HMTAT59  53 720696 T93749, T96255, T69810, T70796, R08624,R08718, R39083, R63142, R70240, R70277, H08586, H08585, R95768, R95815,R99306, R99394, H80069, H80070, N25035, N25050, N27851, N28918, N31176,N31571, N34545, N35790, N40624, N53547, N59046, N63630, N70913, N72886,N80476, W23752, W35196, W57600, W72148, W73164, W73214, W77908,AA025773, AA088588, AA122397, AA122398 44 HDTGC86  54 714037 None 45HAGDI35  55 597444 None 46 HELHN47  56 726157 H16917, R99750, R99927,W37841, AA058809, AA262900 47 HPRBC80  57 829136 N24451, N54675,AA135096, AA164383, AA180531, AA180520, AA179618, AA180509, C17250 47HPRBC80 116 720095 H99661, N24451, N24557, N50019, N54675, AA122002,AA128410, AA164384, AA164383, AA179796, AA179747, AA424173 48 HAQAR23 58 724867 R10492, H72377, H72827, N35006, N74685, W04802, W24689,W90042, W90057, AA085745, AA128131, AA151478, AA149389, AA192154,AA192878 49 HAIFL18  59 676933 None 50 HJPAY76  60 654870 None 51HUSXE77  61 637126 None 52 HUFEF62  62 645101 None 52 HUFEF62 117 630097None 53 HTWJK32  63 699794 N45286, N54734 54 HTWDF76  64 714344 None 55HTPBN68  65 703258 None 56 HTOIY21  66 665745 None 57 HTLDD53  67 626049None 58 HTLFG05  68 879135 AA770231, AA815342, AA854987, AI004529,AI150592, AI200868, AI652314 58 HTLFG05 118 564360 None 59 HDPXR23  69745379 None 59 HDPXR23 119 675684 R24770, R45503, R45503, N67359 60HSIAC45  70 570739 None 61 HSRGW16  71 878849 T95333, T95424, AA470465,AA570056, AA789332, AA682679, AI468303, AI283530, AI634463 61 HSRGW16120 562020 None 62 HSSJC35  72 745409 None 62 HSSJC35 121 716424AA461101 63 HTEAX23  73 609955 None 64 HTGCH22  74 637695 None 65HTJMA95  75 706618 None 66 HHEAA08  76 638231 None 66 HHEAA08 122 623588None 67 HBQAA49  77 598720 None 68 HDPBI32  78 862851 H30751, H38473,H38493, H38543, R85211, R88493, R88620, R88628, R89653, H49766, H51158,W56893, AA604007, AA622175, AA682932, AA984456, AI027311, AI197966 68HDPBI32 123 590733 None 69 HBIBF16  79 580806 None 70 HBCAY05  80 709172R49363, R51623, R49363, R96762, R97690, H98867, N25090, N45327, N52296,N55123, N71799, N72951, W19328, AA120946, AA156710, AA195072, AA195172,AA459898, AA459916, AA417748, AA425926 71 HCUCK44  81 720291 T40562,T91580, T91628, T85780, T98269, R63140, R83013, R83064, R89903, H81296,H81350, H94056, N21020, N21328, N27984, N47889, N51146, N74141, N92869,W04309, W38488, AA043186, AA045597, AA045598, AA074395, AA100050,AA100477, AA100476, AA121548, AA127712, AA130959, AA130829 72 HCE2W56 82 654837 None 73 HCWAG01  83 639022 None 74 HLDBY02  84 587301 None 75HDRMI82  85 877467 T53705, T53428, T53429, T54298, R17396, R42632,R42632, H56472, H87186, H87693, N24851, N29841, N31785, N45025, N57259,N98757, W30988, W68628, AA122061, AA122062, AA122073, AA122027,AA464188, AA464781, AA536154, AA587400, AA610844, AA877474, AA917392,AI031638, C20975, AA651623, F21170, AA861475, AI085701, AI086492,AI086926, AI302609, AI343741, AI356953, AI363021, AI432962, AI129368,AI129882, AI139584, AI189687, AI190062, AI597787, AI312943, AI333509 75HDRMI82 124 654838 None 76 HEPCU48  86 695719 None 77 HDPRK33  87 636059None 77 HDPRK33 125 745378 None 78 HKGAX42  88 517353 None 79 HLMAZ95 89 638588 T60704, T60748, R19381 80 HLMFC07  90 870231 AI382616 80HLMFC07 126 612859 None 81 HL2AG87  91 668244 None 82 HKGCO27  92 601969None 82 HKGCO27 127 581293 None 83 HLDCE79  93 638239 None 83 HLDCE79128 604056 N92706, W20322 84 HERAD40  94 560633 None 85 HFOXB55  95647261 R63343, R63350, R76912, R80129, R80130, H01086, N98518, AA46391986 HFVGZ42  96 634885 T51332, T70321, T70404, T87339, T87440, H47973,H47974, R87191, R87192, R88914, R89592, R89848, R89887, H51778, H57511,H58322, H58527, H59577, H59576, H69658, H70071, H73534, H73535, H73788,H73789, H79203, H91674, H91770, N74679, N77319, N92819, W05041, W20377,W24683, W24998, AA047660, AA243845 87 HNHAF39  97 554908 None 88 HNTSW57 98 861244 T77533, T77569, R73928, H81027, H81122, N21106, N31120,AA195503, AA232666, AA234053, AA459172, AA659810, AA827020, AA837019,AA976790, AI017858, AA187119, AA776129, AI074833, AI040698, AI089608,AI092056, AI092747, AI261793, AI361823 88 HNTSW57 129 638161 None 89HOGCK20  99 745445 R18126, H25231, H46420, H82992, H83225, W32805,AA032233, AA044111 89 HOGCK20 130 664499 None 90 HMDAL49 100 633723 None91 HLYES38 101 638042 None 92 HMECK83 102 636035 None 93 HSHAX21 103612823 None 94 HMQAG66 104 753237 None 94 HMQAG66 131 618856 None

[1253] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLES Example 1 Isolation of a Selected cDNA Clone from the DepositedSample

[1254] Each cDNA clone in a cited ATCC deposit is contained in a plasmidvector. Table 1 identifies the vectors used to construct the cDNAlibrary from which each clone was isolated. In many cases, the vectorused to construct the library is a phage vector from which a plasmid hasbeen excised. The table immediately below correlates the related plasmidfor each phage vector used in constructing the cDNA library. Forexample, where a particular clone is identified in Table 1 as beingisolated in the vector “Lambda Zap,” the corresponding deposited cloneis in “pBluescript.” Vector Used to Construct Library PlasmidCorresponding Deposited Lambda Zap pBluescript (pBS) Uni-Zap XRpBluescript (pBS) Zap Express pBK lafmid BA plafmid BA pSport1 pSport1pCMVSport 2.0 pCMVSport 2.0 pCMVSport 3.0 pCMVSport 3.0 pCR ® 2.1pCR ® 2.1

[1255] Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636),Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S.Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. etal., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. andShort, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees,M. A. et al., Strategies 5:58-61 (1992)) are commercially available fromStratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla,Calif., 92037. pBS contains an ampicillin resistance gene and pBKcontains a neomycin resistance gene. Both can be transformed into E.coli strain XL-1 Blue, also available from Stratagene. pBS comes in 4forms SK+, SK−, KS+ and KS. The S and K refers to the orientation of thepolylinker to the T7 and T3 primer sequences which flank the polylinkerregion (“S” is for SacI and “K” is for KpnI which are the first sites oneach respective end of the linker). “+” or “−” refer to the orientationof the f1 origin of replication (“ori”), such that in one orientation,single stranded rescue initiated from the f1 ori generates sense strandDNA and in the other, antisense.

[1256] Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtainedfrom Life Technologies, Inc., P. O. Box 6009, Gaithersburg, Md. 20897.All Sport vectors contain an ampicillin resistance gene and may betransformed into E. coli strain DH10B, also available from LifeTechnologies. (See, for instance, Gruber, C. E., et al., Focus 15:59(1993).) Vector lafmid BA (Bento Soares, Columbia University, NY)contains an ampicillin resistance gene and can be transformed into E.coli strain XL-1 Blue. Vector pCR®2.1, which is available fromInvitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains anampicillin resistance gene and may be transformed into E. coli strainDH10B, available from Life Technologies. (See, for instance, Clark, J.M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al.,Bio/Technology 9: (1991).) Preferably, a polynucleotide of the presentinvention does not comprise the phage vector sequences identified forthe particular clone in Table 1, as well as the corresponding plasmidvector sequences designated above.

[1257] The deposited material in the sample assigned the ATCC DepositNumber cited in Table 1 for any given cDNA clone also may contain one ormore additional plasmids, each comprising a cDNA clone different fromthat given clone. Thus, deposits sharing the same ATCC Deposit Numbercontain at least a plasmid for each cDNA clone identified in Table 1.Typically, each ATCC deposit sample cited in Table 1 comprises a mixtureof approximately equal amounts (by weight) of about 50 plasmid DNAs,each containing a different cDNA clone; but such a deposit sample mayinclude plasmids for more or less than 50 cDNA clones, up to about 500cDNA clones.

[1258] Two approaches can be used to isolate a particular clone from thedeposited sample of plasmid DNAs cited for that clone in Table 1. First,a plasmid is directly isolated by screening the clones using apolynucleotide probe corresponding to SEQ ID NO:X.

[1259] Particularly, a specific polynucleotide with 3040 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with³²P-γ-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmidmixture is transformed into a suitable host, as indicated above (such asXL-1 Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents cited above. The transformants are plated on1.5% agar plates (containing the appropriate selection agent, e.g.,ampicillin) to a density of about 150 transformants (colonies) perplate. These plates are screened using Nylon membranes according toroutine methods for bacterial colony screening (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold SpringHarbor Laboratory Press, pages 1.93 to 1.104), or other techniques knownto those of skill in the art.

[1260] Alternatively, two primers of 17-20 nucleotides derived from bothends of the SEQ ID NO:X (i.e., within the region of SEQ ID NO:X boundedby the 5′ NT and the 3′ NT of the clone defined in Table 1) aresynthesized and used to amplify the desired cDNA using the depositedcDNA plasmid as a template. The polymerase chain reaction is carried outunder routine conditions, for instance, in 25 ul of reaction mixturewith 0.5 ug of the above cDNA template. A convenient reaction mixture is1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP,dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirtyfive cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at55 degree C. for 1 min; elongation at 72 degree C. for 1 min) areperformed with a Perkin-Elmer Cetus automated thermal cycler. Theamplified product is analyzed by agarose gel electrophoresis and the DNAband with expected molecular weight is excised and purified. The PCRproduct is verified to be the selected sequence by subcloning andsequencing the DNA product.

[1261] Several methods are available for the identification of the 5′ or3′ non-coding portions of a gene which may not be present in thedeposited clone. These methods include but are not limited to, filterprobing, clone enrichment using specific probes, and protocols similaror identical to 5′ and 3′ “RACE” protocols which are well known in theart. For instance, a method similar to 5′ RACE is available forgenerating the missing 5′ end of a desired full-length transcript.(Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993).)

[1262] Briefly, a specific RNA oligonucleotide is ligated to the 5′ endsof a population of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full length gene.

[1263] This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA which may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

[1264] This modified RNA preparation is used as a template for firststrand cDNA synthesis using a gene specific oligonucleotide. The firststrand synthesis reaction is used as a template for PCR amplification ofthe desired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene.

Example 2 Isolation of Genomic Clones Corresponding to a Polynucleotide

[1265] A human genomic P1 library (Genomic Systems, Inc.) is screened byPCR using primers selected for the cDNA sequence corresponding to SEQ IDNO:X., according to the method described in Example 1. (See also,Sambrook.)

Example 3 Tissue Distribution of Polypeptide

[1266] Tissue distribution of mRNA expression of polynucleotides of thepresent invention is determined using protocols for Northern blotanalysis, described by, among others, Sambrook et al. For example, acDNA probe produced by the method described in Example 1 is labeled withp32 using the rediprime™ DNA labeling system (Amersham Life Science),according to manufacturer's instructions. After labeling, the probe ispurified using CHROMA SPIN-100™ column (Clontech Laboratories, Inc.),according to manufacturer's protocol number PT1200-1. The purifiedlabeled probe is then used to examine various human tissues for mRNAexpression.

[1267] Multiple Tissue Northern (MTN) blots containing various humantissues (H) or human immune system tissues (IM) (Clontech) are examinedwith the labeled probe using ExpressHyb™ hybridization solution(Clontech) according to manufacturer's protocol number PT1190-1.Following hybridization and washing, the blots are mounted and exposedto film at −70 degree C. overnight, and the films developed according tostandard procedures.

Example 4 Chromosomal Mapping of the Polynucleotides

[1268] An oligonucleotide primer set is designed according to thesequence at the 5′ end of SEQ ID NO:X. This primer preferably spansabout 100 nucleotides. This primer set is then used in a polymerasechain reaction under the following set of conditions: 30 seconds,95degree C.; 1 minute, 56 degree C.; 1 minute, 70 degree C. This cycle isrepeated 32 times followed by one 5 minute cycle at 70 degree C. Human,mouse, and hamster DNA is used as template in addition to a somatic cellhybrid panel containing individual chromosomes or chromosome fragments(Bios, Inc). The reactions is analyzed on either 8% polyacrylamide gelsor 3.5% agarose gels. Chromosome mapping is determined by the presenceof an approximately 100 bp PCR fragment in the particular somatic cellhybrid.

Example 5 Bacterial Expression of a Polypeptide

[1269] A polynucleotide encoding a polypeptide of the present inventionis amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ ends of the DNA sequence, as outlined in Example 1, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI, at the 5′end of the primers in order to clone the amplified product into theexpression vector. For example, BamHI and XbaI correspond to therestriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Ampr), a bacterial origin of replication (ori),an IPTG-regulatable promoter/operator (P/O), a ribosome binding site(RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

[1270] The pQE-9 vector is digested with BamHI and XbaI and theamplified fragment is ligated into the pQE-9 vector maintaining thereading frame initiated at the bacterial RBS. The ligation mixture isthen used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) whichcontains multiple copies of the plasmid pREP4, which expresses the lacIrepressor and also confers kanamycin resistance (Kan^(r)). Transformantsare identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

[1271] Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(Isopropyl-B-D-thiogalacto pyranoside) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression.

[1272] Cells are grown for an extra 3 to 4 hours. Cells are thenharvested by centrifugation (20 mins at 6000×g). The cell pellet issolubilized in the chaotropic agent 6 Molar Guanidine HCl by stirringfor 3-4 hours at 4 degree C. The cell debris is removed bycentrifugation, and the supernatant containing the polypeptide is loadedonto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

[1273] Briefly, the supernatant is loaded onto the column in 6 Mguanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 Mguanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

[1274] The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4 degree C. or frozen at −80 degree C.

[1275] In addition to the above expression vector, the present inventionfurther includes an expression vector comprising phage operator andpromoter elements operatively linked to a polynucleotide of the presentinvention, called pHE4a. (ATCC Accession Number 209645, deposited onFeb. 25, 1998.) This vector contains:

[1276] 1) a neomycinphosphotransferase gene as a selection marker, 2) anE. coli origin of replication, 3) a T5 phage promoter sequence, 4) twolac operator sequences, 5) a Shine-Delgarno sequence, and 6) the lactoseoperon repressor gene (lacIq). The origin of replication (oriC) isderived from pUC19 (LTI, Gaithersburg, Md.). The promoter sequence andoperator sequences are made synthetically.

[1277] DNA can be inserted into the pHEa by restricting the vector withNdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted product ona gel, and isolating the larger fragment (the stuffer fragment should beabout 310 base pairs). The DNA insert is generated according to the PCRprotocol described in Example 1, using PCR primers having restrictionsites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer).The PCR insert is gel purified and restricted with compatible enzymes.The insert and vector are ligated according to standard protocols.

[1278] The engineered vector could easily be substituted in the aboveprotocol to express protein in a bacterial system.

Example 6 Purification of a Polypeptide from an Inclusion Body

[1279] The following alternative method can be used to purify apolypeptide expressed in E coli when it is present in the form ofinclusion bodies. Unless otherwise specified, all of the following stepsare conducted at 4-10 degree C.

[1280] Upon completion of the production phase of the E. colifermentation, the cell culture is cooled to 4-10 degree C. and the cellsharvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech).On the basis of the expected yield of protein per unit weight of cellpaste and the amount of purified protein required, an appropriate amountof cell paste, by weight, is suspended in a buffer solution containing100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to ahomogeneous suspension using a high shear mixer.

[1281] The cells are then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

[1282] The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCl extraction.

[1283] Following high speed centrifugation (30,000×g) to removeinsoluble particles, the GuHCl solubilized protein is refolded byquickly mixing the GuHCl extract with 20 volumes of buffer containing 50mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. Therefolded diluted protein solution is kept at 4 degree C. without mixingfor 12 hours prior to further purification steps.

[1284] To clarify the refolded polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 um membranefilter with appropriate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 nm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

[1285] Fractions containing the polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the polypeptide (determined, for instance, by 16% SDS-PAGE)are then pooled.

[1286] The resultant polypeptide should exhibit greater than 95% purityafter the above refolding and purification steps. No major contaminantbands should be observed from Commassie blue stained 16% SDS-PAGE gelwhen 5 ug of purified protein is loaded. The purified protein can alsobe tested for endotoxin/LPS contamination, and typically the LPS contentis less than 0.1 ng/ml according to LAL assays.

Example 7 Cloning and Expression of a Polypeptide in a BaculovirusExpression System

[1287] In this example, the plasmid shuttle vector pA2 is used to inserta polynucleotide into a baculovirus to express a polypeptide. Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI, Xba I and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate a viable virus thatexpress the cloned polynucleotide.

[1288] Many other baculovirus vectors can be used in place of the vectorabove, such as pAc373, pVL941, and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (1989).

[1289] Specifically, the cDNA sequence contained in the deposited clone,including the AUG initiation codon and the naturally associated leadersequence identified in Table 1, is amplified using the PCR protocoldescribed in Example 1. If the naturally occurring signal sequence isused to produce the secreted protein, the pA2 vector does not need asecond signal peptide. Alternatively, the vector can be modified (pA2GP) to include a baculovirus leader sequence, using the standard methodsdescribed in Summers et al., “A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures,” Texas AgriculturalExperimental Station Bulletin No. 1555 (1987).

[1290] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

[1291] The plasmid is digested with the corresponding restrictionenzymes and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.).

[1292] The fragment and the dephosphorylated plasmid are ligatedtogether with T4 DNA ligase. E. coli HB101 or other suitable E. colihosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria containing the plasmid are identified by digesting DNAfrom individual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

[1293] Five ug of a plasmid containing the polynucleotide isco-transfected with 1.0 ug of a commercially available linearizedbaculovirus DNA (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego,Calif.), using the lipofection method described by Felgner et al., Proc.Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virusDNA and 5 ug of the plasmid are mixed in a sterile well of a microtiterplate containing 50 ul of serum-free Grace's medium (Life TechnologiesInc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ulGrace's medium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

[1294] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10.) After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

[1295] To verify the expression of the polypeptide, Sf9 cells are grownin Grace's medium supplemented with 10% heat-inactivated FBS. The cellsare infected with the recombinant baculovirus containing thepolynucleotide at a multiplicity of infection (“MOI”) of about 2. Ifradiolabeled proteins are desired, 6 hours later the medium is removedand is replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). After 42 hours,5 uCi of ³⁵S-methionine and 5 uCi ³⁵S-cysteine (available from Amersham)are added. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

[1296] Microsequencing of the amino acid sequence of the amino terminusof purified protein may be used to determine the amino terminal sequenceof the produced protein.

Example 8 Expression of a Polypeptide in Mammalian Cells

[1297] The polypeptide of the present invention can be expressed in amammalian cell. A typical mammalian expression vector contains apromoter element, which mediates the initiation of transcription ofmRNA, a protein coding sequence, and signals required for thetermination of transcription and polyadenylation of the transcript.Additional elements include enhancers, Kozak sequences and interveningsequences flanked by donor and acceptor sites for RNA splicing. Highlyefficient transcription is achieved with the early and late promotersfrom SV40, the long terminal repeats (LTRs) from Retroviruses, e.g.,RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter).

[1298] Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0.Mammalian host cells that could be used include, human Hela, 293, H9 andJurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quailQC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

[1299] Alternatively, the polypeptide can be expressed in stable celllines containing the polynucleotide integrated into a chromosome. Theco-transfection with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transfectedcells.

[1300] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful in developing cell lines that carry several hundred oreven several thousand copies of the gene of interest. (See, e.g., Alt,F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. andMa, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. andSydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selectionmarker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992).Using these markers, the mammalian cells are grown in selective mediumand the cells with the highest resistance are selected. These cell linescontain the amplified gene(s) integrated into a chromosome. Chinesehamster ovary (CHO) and NSO cells are often used for the production ofproteins.

[1301] Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146),the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCCAccession No.209647) contain the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985).) Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors also contain the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene,and the mouse DHFR gene under control of the SV40 early promoter.

[1302] Specifically, the plasmid pC6, for example, is digested withappropriate restriction enzymes and then dephosphorylated using calfintestinal phosphates by procedures known in the art. The vector is thenisolated from a 1% agarose gel.

[1303] A polynucleotide of the present invention is amplified accordingto the protocol outlined in Example 1. If the naturally occurring signalsequence is used to produce the secreted protein, the vector does notneed a second signal peptide. Alternatively, if the naturally occurringsignal sequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/34891.)

[1304] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

[1305] The amplified fragment is then digested with the same restrictionenzyme and purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 using,for instance, restriction enzyme analysis.

[1306] Chinese hamster ovary cells lacking an active DHFR gene is usedfor transfection. Five μg of the expression plasmid pC6 a pC4 iscotransfected with 0.5 ug of the plasmid pSVneo using lipofectin(Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the neo gene from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days singleclones are trypsinized and then seeded in 6-well petri dishes or 10 mlflasks using different concentrations of methotrexate (50 nM, 100 nM,200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM).The same procedure is repeated until clones are obtained which grow at aconcentration of 100-200 uM. Expression of the desired gene product isanalyzed, for instance, by SDS-PAGE and Western blot or by reversedphase HPLC analysis.

Example 9 Protein Fusions

[1307] The polypeptides of the present invention are preferably fused toother proteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Example 5; see also EP A 394,827;Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion toIgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclearlocalization signals fused to the polypeptides of the present inventioncan target the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof a fusion protein. Fusion proteins can also create chimeric moleculeshaving more than one function. Finally, fusion proteins can increasesolubility and/or stability of the fused protein compared to thenon-fused protein. All of the types of fusion proteins described abovecan be made by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule, or the protocol described inExample 5.

[1308] Briefly, the human Fc portion of the IgG molecule can be PCRamplified, using primers that span the 5′ and 3′ ends of the sequencedescribed below. These primers also should have convenient restrictionenzyme sites that will facilitate cloning into an expression vector,preferably a mammalian expression vector.

[1309] For example, if pC4 (Accession No. 209646) is used, the human Fcportion can be ligated into the BamHI cloning site. Note that the 3′BamHI site should be destroyed. Next, the vector containing the human Fcportion is re-restricted with BamFHi, linearizing the vector, and apolynucleotide of the present invention, isolated by the PCR protocoldescribed in Example 1, is ligated into this BamHI site. Note that thepolynucleotide is cloned without a stop codon, otherwise a fusionprotein will not be produced.

[1310] If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

[1311] Human IgG Fc Region:

[1312] To construct a synthetic GAS containing promoter element, whichis used in the Biological Assays described in Examples 13-14, a PCRbased strategy is employed to generate a GAS-SV40 promoter sequence. The5′ primer contains four tandem copies of the GAS binding site found inthe IRF1 promoter and previously demonstrated to bind STATs uponinduction with a range of cytokines (Rothman et al., Immunity 1:457-468(1994).), although other GAS or ISRE elements can be used instead. The5′ primer also contains 18 bp of sequence complementary to the SV40early promoter sequence and is flanked with an XhoI site. The sequenceof the 5′ primer is: 5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCC (SEQ ID NO: 3)GAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG: 3′

[1313] The downstream primer is complementary to the SV40 promoter andis flanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQID NO:4)

[1314] PCR amplification is performed using the SV40 promoter templatepresent in the B-gal:promoter plasmid obtained from Clontech. Theresulting PCR fragment is digested with XhoI/Hind III and subcloned intoBLSK2−. (Stratagene.) Sequencing with forward and reverse primersconfirms that the insert contains the following sequence: 5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAA (SEQ ID NO: 5)TGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTA GGCTTTTGCAAAAAGCTT:3′

[1315] With this GAS promoter element linked to the SV40 promoter, aGAS:SEAP2 reporter construct is next engineered. Here, the reportermolecule is a secreted alkaline phosphatase, or “SEAP.” Clearly,however, any reporter molecule can be instead of SEAP, in this or in anyof the other Examples. Well known reporter molecules that can be usedinstead of SEAP include chloramphenicol acetyltransferase (CAT),luciferase, alkaline phosphatase, B-galactosidase, green fluorescentprotein (GFP), or any protein detectable by an antibody.

[1316] The above sequence confirmed synthetic GAS-SV40 promoter elementis subcloned into the pSEAP-Promoter vector obtained from Clontech usingHindIII and XhoI, effectively replacing the SV40 promoter with theamplified GAS:SV40 promoter element, to create the GAS-SEAP vector.However, this vector does not contain a neomycin resistance gene, andtherefore, is not preferred for mammalian expression systems.

[1317] Thus, in order to generate mammalian stable cell lines expressingthe GAS-SEAP reporter, the GAS-SEAP cassette is removed from theGAS-SEAP vector using SalI and NotI, and inserted into a backbone vectorcontaining the neomycin resistance gene, such as pGFP-1 (Clontech),using these restriction sites in the multiple cloning site, to createthe GAS-SEAP/Neo vector. Once this vector is transfected into mammaliancells, this vector can then be used as a reporter molecule for GASbinding as described in Examples 13-14.

[1318] Other constructs can be made using the above description andreplacing GAS with a different promoter sequence. For example,construction of reporter molecules containing NFK-B and EGR promotersequences are described in Examples 15 and 16. However, many otherpromoters can be substituted using the protocols described in theseExamples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can besubstituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB,Il-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used totest reporter construct activity, such as HELA (epithelial), HUVEC(endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), orCardiomyocyte.

Example 13 High-Throughput Screening Assay for T-cell Activity

[1319] The following protocol is used to assess T-cell activity byidentifying factors, and determining whether supernate containing apolypeptide of the invention proliferates and/or differentiates T-cells.T-cell activity is assessed using the GAS/SEAP/Neo construct produced inExample 12. Thus, factors that increase SEAP activity indicate theability to activate the Jaks-STATS signal transduction pathway. TheT-cell used in this assay is Jurkat T-cells (ATCC Accession No.TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4cells (ATCC Accession No. CRL-1582) cells can also be used.

[1320] Jurkat T-cells are lymphoblastic CD4⁺ Th1 helper cells. In orderto generate stable cell lines, approximately 2 million Jurkat cells aretransfected with the GAS-SEAP/neo vector using DMRIE-C (LifeTechnologies)(transfection procedure described below). The transfectedcells are seeded to a density of approximately 20,000 cells per well andtransfectants resistant to 1 mg/ml genticin selected. Resistant coloniesare expanded and then tested for their response to increasingconcentrations of interferon gamma. The dose response of a selectedclone is demonstrated.

[1321] Specifically, the following protocol will yield sufficient cellsfor 75 wells containing 200 ul of cells. Thus, it is either scaled up,or performed in multiple to generate sufficient cells for multiple 96well plates. Jurkat cells are maintained in RPMI+10% serum with 1%Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug ofplasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul ofDMRIE-C and incubate at room temperature for 15-45 mins.

[1322] During the incubation period, count cell concentration, spin downthe required number of cells (10⁷ per transfection), and resuspend inOPTI-MEM to a final concentration of 10⁷ cells/ml. Then add 1 ml of1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37 degrees C. for 6hrs. After the incubation, add 10 ml of RPMI+15% serum.

[1323] The Jurkat:GAS-SEAP stable reporter lines are maintained inRPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells aretreated with supernatants containing polypeptides of the inventionand/or induced polypeptides of the invention as produced by the protocoldescribed in Example 11.

[1324] On the day of treatment with the supernatant, the cells should bewashed and resuspended in fresh RPMI+10% serum to a density of 500,000cells per ml. The exact number of cells required will depend on thenumber of supernatants being screened. For one 96 well plate,approximately 10 million cells (for 10 plates, 100 million cells) arerequired.

[1325] Transfer the cells to a triangular reservoir boat, in order todispense the cells into a 96 well dish, using a 12 channel pipette.Using a 12 channel pipette, transfer 200 ul of cells into each well(therefore adding 100,000 cells per well).

[1326] After all the plates have been seeded, 50 ul of the supernatantsare transferred directly from the 96 well plate containing thesupernatants into each well using a 12 channel pipette. In addition, adose of exogenous interferon gamma (0.1, 1.0, 10 ng) is added to wellsH9, H10, and H11 to serve as additional positive controls for the assay.

[1327] The 96 well dishes containing Jurkat cells treated withsupernatants are placed in an incubator for 48 hrs (note: this time isvariable between 48-72 hrs). 35 ul samples from each well are thentransferred to an opaque 96 well plate using a 12 channel pipette. Theopaque plates should be covered (using sellophene covers) and stored at−20 degrees C. until SEAP assays are performed according to Example 17.The plates containing the remaining treated cells are placed at 4degrees C. and serve as a source of material for repeating the assay ona specific well if desired.

[1328] As a positive control, 100 Unit/ml interferon gamma can be usedwhich is known to activate Jurkat T cells. Over 30 fold induction istypically observed in the positive control wells.

[1329] The above protocol may be used in the generation of bothtransient, as well as, stable transfected cells, which would be apparentto those of skill in the art.

Example 14 High-Throughput Screening Assay Identifying Myeloid Activity

[1330] The following protocol is used to assess myeloid activity bydetermining whether polypeptides of the invention proliferates and/ordifferentiates myeloid cells. Myeloid cell activity is assessed usingthe GAS/SEAP/Neo construct produced in Example 12. Thus, factors thatincrease SEAP activity indicate the ability to activate the Jaks-STATSsignal transduction pathway. The myeloid cell used in this assay isU937, a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used.

[1331] To transiently transfect U937 cells with the GAS/SEAP/Neoconstruct produced in Example 12, a DEAE-Dextran method (Kharbanda et.al., 1994, Cell Growth & Differentiation, 5:259-265) is used. First,harvest 2×10e⁷ U937 cells and wash with PBS. The U937 cells are usuallygrown in RPMI 1640 medium containing 10% heat-inactivated fetal bovineserum (FBS) supplemented with 100 units/ml penicillin and 100 mg/mlstreptomycin.

[1332] Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffercontaining 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mMNaCl, 5 mM KCl, 375 uM Na₂HPO₄.7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂.Incubate at 37 degrees C. for 45 min.

[1333] Wash the cells with RPMI 1640 medium containing 10% FBS and thenresuspend in 10 ml complete medium and incubate at 37 degrees C. for 36hr.

[1334] The GAS-SEAP/U937 stable cells are obtained by growing the cellsin 400 ug/ml G418. The G418-free medium is used for routine growth butevery one to two months, the cells should be re-grown in 400 ug/ml G418for couple of passages.

[1335] These cells are tested by harvesting 1×10⁸ cells (this is enoughfor ten 96-well plates assay) and wash with PBS. Suspend the cells in200 ml above described growth medium, with a final density of 5×10⁵cells/ml. Plate 200 ul cells per well in the 96-well plate (or 1×10⁵cells/well).

[1336] Add 50 ul of the supernatant prepared by the protocol describedin Example 11. Incubate at 37 degrees C. for 48 to 72 hr. As a positivecontrol, 100 Unit/ml interferon gamma can be used which is known toactivate U937 cells. Over 30 fold induction is typically observed in thepositive control wells. SEAP assay the supernatant according to theprotocol described in Example 17.

Example 15 High-Throughput Screening Assay Identifying Neuronal Activity

[1337] When cells undergo differentiation and proliferation, a group ofgenes are activated through many different signal transduction pathways.One of these genes, EGR1 (early growth response gene 1), is induced invarious tissues and cell types upon activation. The promoter of EGR1 isresponsible for such induction. Using the EGR1 promoter linked toreporter molecules, activation of cells can be assessed.

[1338] Particularly, the following protocol is used to assess neuronalactivity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells)are known to proliferate and/or differentiate by activation with anumber of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF(nerve growth factor), and EGF (epidermal growth factor). The EGR1 geneexpression is activated during this treatment. Thus, by stablytransfecting PC12 cells with a construct containing an EGR promoterlinked to SEAP reporter, activation of PC12 cells can be assessed.

[1339] The EGR/SEAP reporter construct can be assembled by the followingprotocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al.,Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNAusing the following primers: 5′ GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′ (SEQID NO: 6) 5′ GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′ (SEQ ID NO: 7)

[1340] Using the GAS:SEAP/Neo vector produced in Example 12, EGR1amplified product can then be inserted into this vector. Linearize theGAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing theGAS/SV40 stuffer. Restrict the EGR1 amplified product with these sameenzymes. Ligate the vector and the EGR1 promoter.

[1341] To prepare 96 well-plates for cell culture, two mls of a coatingsolution (1:30 dilution of collagen type I (Upstate Biotech Inc.Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cmplate or 50 ml per well of the 96-well plate, and allowed to air dry for2 hr.

[1342] PC12 cells are routinely grown in RPMI-1640 medium (BioWhittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. #12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplementedwith 100 units/ml penicillin and 100 ug/ml streptomycin on a precoated10 cm tissue culture dish. One to four split is done every three to fourdays. Cells are removed from the plates by scraping and resuspended withpipetting up and down for more than 15 times.

[1343] Transfect the EGR/SEAP/Neo construct into PC12 using theLipofectamine protocol described in Example 11. EGR-SEAP/PC12 stablecells are obtained by growing the cells in 300 ug/ml G418. The G418-freemedium is used for routine growth but every one to two months, the cellsshould be re-grown in 300 ug/ml G418 for couple of passages.

[1344] To assay for neuronal activity, a 10 cm plate with cells around70 to 80% confluent is screened by removing the old medium. Wash thecells once with PBS (Phosphate buffered saline). Then starve the cellsin low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBSwith antibiotics) overnight.

[1345] The next morning, remove the medium and wash the cells with PBS.Scrape off the cells from the plate, suspend the cells well in 2 ml lowserum medium. Count the cell number and add more low serum medium toreach final cell density as 5×10⁵ cells/ml.

[1346] Add 200 ul of the cell suspension to each well of 96-well plate(equivalent to 1×10⁵ cells/well). Add 50 ul supernatant produced byExample 11, 37° C. for 48 to 72 hr. As a positive control, a growthfactor known to activate PC12 cells through EGR can be used, such as 50ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAPis typically seen in the positive control wells. SEAP assay thesupernatant according to Example 17.

Example 16 High-Throughput Screening Assay for T-cell Activity

[1347] NF-KB (Nuclear Factor KB) is a transcription factor activated bya wide variety of agents including the inflammatory cytokines IL-1 andTNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposureto LPS or thrombin, and by expression of certain viral gene products. Asa transcription factor, NF-KB regulates the expression of genes involvedin immune cell activation, control of apoptosis (NF-KB appears to shieldcells from apoptosis), B and T-cell development, anti-viral andantimicrobial responses, and multiple stress responses.

[1348] In non-stimulated conditions, NF-KB is retained in the cytoplasmwith I-KB (Inhibitor KB). However, upon stimulation, I-KB isphosphorylated and degraded, causing NF-KB to shuttle to the nucleus,thereby activating transcription of target genes. Target genes activatedby NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

[1349] Due to its central role and ability to respond to a range ofstimuli, reporter constructs utilizing the NF-KB promoter element areused to screen the supernatants produced in Example 11. Activators orinhibitors of NF-KB would be useful in treating diseases. For example,inhibitors of NF-KB could be used to treat those diseases related to theacute or chronic activation of NF-KB, such as rheumatoid arthritis.

[1350] To construct a vector containing the NF-KB promoter element, aPCR based strategy is employed. The upstream primer contains four tandemcopies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO:8), 18 bp ofsequence complementary to the 5′ end of the SV40 early promotersequence, and is flanked with an XhoI site: 5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGAC (SEQ ID NO: 9)TTTCCATCCTGCCATCTCAATTAG: 3′

[1351] The downstream primer is complementary to the 3′ end of the SV40promoter and is flanked with a Hind III site:5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO:4)

[1352] PCR amplification is performed using the SV40 promoter templatepresent in the pB-gal:promoter plasmid obtained from Clontech. Theresulting PCR fragment is digested with XhoI and Hind III and subclonedinto BLSK2−. (Stratagene) Sequencing with the T7 and T3 primers confirmsthe insert contains the following sequence: 5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCC (SEQ ID NO: 10)ATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAA GCTT: 3′

[1353] Next, replace the SV40 minimal promoter element present in thepSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment usingXhoI and HindIII. However, this vector does not contain a neomycinresistance gene, and therefore, is not preferred for mammalianexpression systems.

[1354] In order to generate stable mammalian cell lines, theNF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP vectorusing restriction enzymes SalI and NotI, and inserted into a vectorcontaining neomycin resistance. Particularly, the NF-KB/SV40/SEAPcassette was inserted into pGFP-1 (Clontech), replacing the GFP gene,after restricting pGFP-1 with SalI and NotI.

[1355] Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cellsare created and maintained according to the protocol described inExample 13. Similarly, the method for assaying supernatants with thesestable Jurkat T-cells is also described in Example 13. As a positivecontrol, exogenous TNF alpha (0.1, 1, 10 ng) is added to wells H9, H10,and H11, with a 5-10 fold activation typically observed.

Example 17 Assay for SEAP Activity

[1356] As a reporter molecule for the assays described in Examples13-16, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat.BP-400) according to the following general procedure. The TropixPhospho-light Kit supplies the Dilution, Assay, and Reaction Buffersused below.

[1357] Prime a dispenser with the 2.5× Dilution Buffer and dispense 15ul of 2.5× dilution buffer into Optiplates containing 35 ul of asupernatant. Seal the plates with a plastic sealer and incubate at 65degree C. for 30 min. Separate the Optiplates to avoid uneven heating.

[1358] Cool the samples to room temperature for 15 minutes. Empty thedispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer andincubate at room temperature 5 min. Empty the dispenser and prime withthe Reaction Buffer (see the table below). Add 50 ul Reaction Buffer andincubate at room temperature for 20 minutes. Since the intensity of thechemiluminescent signal is time dependent, and it takes about 10 minutesto read 5 plates on luminometer, one should treat 5 plates at each timeand start the second set 10 minutes later.

[1359] Read the relative light unit in the luminometer. Set H12 asblank, and print the results. An increase in chemiluminescence indicatesreporter activity. Reaction Buffer Formulation: # of plates Rxn bufferdiluent (ml) CSPD (ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 415 85 4.25 16 90 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 1155.75 22 120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28150 7.5 29 155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 935 185 9.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41215 10.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47245 12.25 48 250 12.5 49 255 12.75 50 260 13

Example 18 High-Throughput Screening Assay Identifying Changes in SmallMolecule Concentration and Membrane Permeability

[1360] Binding of a ligand to a receptor is known to alter intracellularlevels of small molecules, such as calcium, potassium, sodium, and pH,as well as alter membrane potential. These alterations can be measuredin an assay to identify supernatants which bind to receptors of aparticular cell. Although the following protocol describes an assay forcalcium, this protocol can easily be modified to detect changes inpotassium, sodium, pH, membrane potential, or any other small moleculewhich is detectable by a fluorescent probe.

[1361] The following assay uses Fluorometric Imaging Plate Reader(“FLIPR”) to measure changes in fluorescent molecules (Molecular Probes)that bind small molecules. Clearly, any fluorescent molecule detecting asmall molecule can be used instead of the calcium fluorescent molecule,fluo-4 (Molecular Probes, Inc.; catalog no. F-14202), used here.

[1362] For adherent cells, seed the cells at 10,000-20,000 cells/well ina Co-star black 96-well plate with clear bottom. The plate is incubatedin a CO₂ incubator for 20 hours. The adherent cells are washed two timesin Biotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution)leaving 100 ul of buffer after the final wash.

[1363] A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acidDMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is addedto each well. The plate is incubated at 37 degrees C. in a CO₂ incubatorfor 60 min. The plate is washed four times in the Biotek washer with BSSleaving 100 ul of buffer.

[1364] For non-adherent cells, the cells are spun down from culturemedia. Cells are re-suspended to 2-5×10⁶ cells/ml with BSS in a 50-mlconical tube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSOis added to each ml of cell suspension. The tube is then placed in a 37degrees C. water bath for 30-60 min. The cells are washed twice withHBSS, resuspended to 1×10⁶ cells/ml, and dispensed into a microplate,100 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plateis then washed once in Denley CellWash with 200 ul, followed by anaspiration step to 100 ul final volume.

[1365] For a non-cell based assay, each well contains a fluorescentmolecule, such as fluo-4. The supernatant is added to the well, and achange in fluorescence is detected.

[1366] To measure the fluorescence of intracellular calcium, the FLIPRis set for the following parameters: (1) System gain is 300-800 mW; (2)Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul.Increased emission at 530 nm indicates an extracellular signaling eventwhich has resulted in an increase in the intracellular Ca⁺⁺concentration.

Example 19 High-Throughput Screening Assay Identifying Tyrosine KinaseActivity

[1367] The Protein Tyrosine Kinases (PTK) represent a diverse group oftransmembrane and cytoplasmic kinases. Within the Receptor ProteinTyrosine Kinase RPTK) group are receptors for a range of mitogenic andmetabolic growth factors including the PDGF, FGF, EGF, NGF, HGF andInsulin receptor subfamilies. In addition there are a large family ofRPTKs for which the corresponding ligand is unknown. Ligands for RPTKsinclude mainly secreted small proteins, but also membrane-bound andextracellular matrix proteins.

[1368] Activation of RPTK by ligands involves ligand-mediated receptordimerization, resulting in transphosphorylation of the receptor subunitsand activation of the cytoplasmic tyrosine kinases. The cytoplasmictyrosine kinases include receptor associated tyrosine kinases of thesrc-family (e.g., src, yes, Ick, lyn, fyn) and non-receptor linked andcytosolic protein tyrosine kinases, such as the Jak family, members ofwhich mediate signal transduction triggered by the cytokine superfamilyof receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

[1369] Because of the wide range of known factors capable of stimulatingtyrosine kinase activity, the identification of novel human secretedproteins capable of activating tyrosine kinase signal transductionpathways are of interest. Therefore, the following protocol is designedto identify those novel human secreted proteins capable of activatingthe tyrosine kinase signal transduction pathways.

[1370] Seed target cells (e.g., primary keratinocytes) at a density ofapproximately 25,000 cells per well in a 96 well Loprodyne Silent ScreenPlates purchased from Nalge Nunc (Naperville, Ill.). The plates aresterilized with two 30 minute rinses with 100% ethanol, rinsed withwater and dried overnight. Some plates are coated for 2 hr with 100 mlof cell culture grade type I collagen (50 mg/ml), gelatin (2%) orpolylysine (50 mg/ml), all of which can be purchased from SigmaChemicals (St. Louis, Mo.) or 10% Matrigel purchased from BectonDickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at4 degree C. Cell growth on these plates is assayed by seeding 5,000cells/well in growth medium and indirect quantitation of cell numberthrough use of alamarBlue as described by the manufacturer AlamarBiosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers#3071 from Becton Dickinson (Bedford, Mass.) are used to cover theLoprodyne Silent Screen Plates. Falcon Microtest III cell culture platescan also be used in some proliferation experiments.

[1371] To prepare extracts, A431 cells are seeded onto the nylonmembranes of Loprodyne plates (20,000/200 ml/well) and culturedovernight in complete medium. Cells are quiesced by incubation inserum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF(60 ng/ml) or 50 ul of the supernatant produced in Example 11, themedium was removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5,0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P207 and acocktail of protease inhibitors (# 1836170) obtained from BoeheringerMannheim (Indianapolis, Ind.) is added to each well and the plate isshaken on a rotating shaker for 5 minutes at 4 degrees C. The plate isthen placed in a vacuum transfer manifold and the extract filteredthrough the 0.45 mm membrane bottoms of each well using house vacuum.Extracts are collected in a 96-well catch/assay plate in the bottom ofthe vacuum manifold and immediately placed on ice. To obtain extractsclarified by centrifugation, the content of each well, after detergentsolubilization for 5 minutes, is removed and centrifuged for 15 minutesat 4 degrees C. at 16,000×g.

[1372] Test the filtered extracts for levels of tyrosine kinaseactivity. Although many methods of detecting tyrosine kinase activityare known, one method is described here.

[1373] Generally, the tyrosine kinase activity of a supernatant isevaluated by determining its ability to phosphorylate a tyrosine residueon a specific substrate (a biotinylated peptide). Biotinylated peptidesthat can be used for this purpose include PSK1 (corresponding to aminoacids 6-20 of the cell division kinase cdc2-p34) and PSK2 (correspondingto amino acids 1-17 of gastrin). Both peptides are substrates for arange of tyrosine kinases and are available from Boehringer Mannheim.

[1374] The tyrosine kinase reaction is set up by adding the followingcomponents in order. First, add 10 ul of 5 uM Biotinylated Peptide, then10 ul ATP/Mg₂₊ (5 mM ATP/50 mM MgCl₂), then 10 ul of 5× Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mMEGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then 5 ul of SodiumVanadate(1 mM), and then 5 ul of water. Mix the components gently andpreincubate the reaction mix at 30 degrees C. for 2 min. Initial thereaction by adding 10 ul of the control enzyme or the filteredsupernatant.

[1375] The tyrosine kinase assay reaction is then terminated by adding10 ul of 120 mm EDTA and place the reactions on ice.

[1376] Tyrosine kinase activity is determined by transferring 50 ulaliquot of reaction mixture to a microtiter plate (MTP) module andincubating at 37 degrees C. for 20 min. This allows the streptavadincoated 96 well plate to associate with the biotinylated peptide. Washthe MTP module with 300 ul/well of PBS four times. Next add 75 ul ofanti-phospotyrosine antibody conjugated to horse radishperoxidase(anti-P-Tyr-POD(0.5 u/ml)) to each well and incubate at 37degrees C. for one hour. Wash the well as above.

[1377] Next add 100 ul of peroxidase substrate solution (BoehringerMannheim) and incubate at room temperature for at least 5 mins (up to 30min). Measure the absorbance of the sample at 405 nm by using ELISAreader. The level of bound peroxidase activity is quantitated using anELISA reader and reflects the level of tyrosine kinase activity.

Example 20 High-Throughput Screening Assay Identifying PhosphorylationActivity

[1378] As a potential alternative and/or compliment to the assay ofprotein tyrosine kinase activity described in Example 19, an assay whichdetects activation (phosphorylation) of major intracellular signaltransduction intermediates can also be used. For example, as describedbelow one particular assay can detect tyrosine phosphorylation of theErk-1 and Erk-2 kinases. However, phosphorylation of other molecules,such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src,Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as anyother phosphoserine, phosphotyrosine, or phosphothreonine molecule, canbe detected by substituting these molecules for Erk-1 or Erk-2 in thefollowing assay.

[1379] Specifically, assay plates are made by coating the wells of a96-well ELISA plate with 0.1 ml of protein G (lug/ml) for 2 hr at roomtemp, (RT). The plates are then rinsed with PBS and blocked with 3%BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2commercial monoclonal antibodies (10 ng/well) against Erk-land Erk-2 (1hr at RT) (Santa Cruz Biotechnology). (To detect other molecules, thisstep can easily be modified by substituting a monoclonal antibodydetecting any of the above described molecules.) After 3-5 rinses withPBS, the plates are stored at 4 degrees C. until use.

[1380] A431 cells are seeded at 20,000/well in a 96-well Loprodynefilter plate and cultured overnight in growth medium. The cells are thenstarved for 48 hr in basal medium (DMEM) and then treated with EGF (6ng/well) or 50 ul of the supernatants obtained in Example 11 for 5-20minutes. The cells are then solubilized and extracts filtered directlyinto the assay plate.

[1381] After incubation with the extract for 1 hr at RT, the wells areagain rinsed. As a positive control, a commercial preparation of MAPkinase (10 ng/well) is used in place of A431 extract. Plates are thentreated with a commercial polyclonal (rabbit) antibody (lug/ml) whichspecifically recognizes the phosphorylated epitope of the Erk-1 andErk-2 kinases (1 hr at RT). This antibody is biotinylated by standardprocedures. The bound polyclonal antibody is then quantitated bysuccessive incubations with Europium-streptavidin and Europiumfluorescence enhancing reagent in the Wallac DELFIA instrument(time-resolved fluorescence). An increased fluorescent signal overbackground indicates a phosphorylation.

Example 21 Method of Determining Alterations in a Gene Corresponding toa Polynucleotide

[1382] RNA isolated from entire families or individual patientspresenting with a phenotype of interest (such as a disease) is beisolated. cDNA is then generated from these RNA samples using protocolsknown in the art. (See, Sambrook.) The cDNA is then used as a templatefor PCR, employing primers surrounding regions of interest in SEQ IDNO:X. Suggested PCR conditions consist of 35 cycles at 95 degrees C. for30 seconds; 60-120 seconds at 52-58 degrees C.; and 60-120 seconds at 70degrees C., using buffer solutions described in Sidransky et al.,Science 252:706 (1991).

[1383] PCR products are then sequenced using primers labeled at their 5′end with T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons isalso determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations is then cloned andsequenced to validate the results of the direct sequencing.

[1384] PCR products is cloned into T-tailed vectors as described inHolton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced withT7 polymerase (United States Biochemical). Affected individuals areidentified by mutations not present in unaffected individuals.

[1385] Genomic rearrangements are also observed as a method ofdetermining alterations in a gene corresponding to a polynucleotide.Genomic clones isolated according to Example 2 are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson et al., Methods Cell Biol. 35:73-99(1991). Hybridization with the labeled probe is carried out using a vastexcess of human cot-i DNA for specific hybridization to thecorresponding genomic locus.

[1386] Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region hybridized by the probe are identifiedas insertions, deletions, and translocations. These alterations are usedas a diagnostic marker for an associated disease.

Example 22 Method of Detecting Abnormal Levels of a Polypeptide in aBiological Sample

[1387] A polypeptide of the present invention can be detected in abiological sample, and if an increased or decreased level of thepolypeptide is detected, this polypeptide is a marker for a particularphenotype. Methods of detection are numerous, and thus, it is understoodthat one skilled in the art can modify the following assay to fit theirparticular needs.

[1388] For example, antibody-sandwich ELISAs are used to detectpolypeptides in a sample, preferably a biological sample. Wells of amicrotiter plate are coated with specific antibodies, at a finalconcentration of 0.2 to 10 ug/ml. The antibodies are either monoclonalor polyclonal and are produced byd the method described in

Example 10 The wells are blocked so that non-specific binding of thepolypeptide to the well is reduced.

[1389] The coated wells are then incubated for >2 hours at RT with asample containing the polypeptide. Preferably, serial dilutions of thesample should be used to validate results. The plates are then washedthree times with deionized or distilled water to remove unboundedpolypeptide.

[1390] Next, 50 ul of specific antibody-alkaline phosphatase conjugate,at a concentration of 25-400 ng, is added and incubated for 2 hours atroom temperature. The plates are again washed three times with deionizedor distilled water to remove unbounded conjugate.

[1391] Add 75 ul of 4-methylumbelliferyl phosphate (MUP) orp-nitrophenyl phosphate (NPP) substrate solution to each well andincubate 1 hour at room temperature. Measure the reaction by amicrotiter plate reader. Prepare a standard curve, using serialdilutions of a control sample, and plot polypeptide concentration on theX-axis (log scale) and fluorescence or absorbance of the Y-axis (linearscale). Interpolate the concentration of the polypeptide in the sampleusing the standard curve.

Example 23 Formulation

[1392] The invention also provides methods of treatment and/orprevention of diseases or disorders (such as, for example, any one ormore of the diseases or disorders disclosed herein) by administration toa subject of an effective amount of a Therapeutic. By therapeutic ismeant a polynucleotides or polypeptides of the invention (includingfragments and variants), agonists or antagonists thereof, and/orantibodies thereto, in combination with a pharmaceutically acceptablecarrier type (e.g., a sterile carrier).

[1393] The Therapeutic will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with the Therapeutic alone), the site of delivery, the methodof administration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

[1394] As a general proposition, the total pharmaceutically effectiveamount of the Therapeutic administered parenterally per dose will be inthe range of about lug/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the Therapeutic is typicallyadministered at a dose rate of about 1 ug/kg/hour to about 50ug/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

[1395] Therapeutics can be are administered orally, rectally,parenterally, intracistemally, intravaginally, intraperitoneally,topically (as by powders, ointments, gels, drops or transdermal patch),bucally, or as an oral or nasal spray.

[1396] “Pharmaceutically acceptable carrier” refers to a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitondeal, intrasternal, subcutaneous andintraarticular injection and infusion.

[1397] Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[1398] Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

[1399] Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

[1400] Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317-327 and 353-365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalTherapeutic.

[1401] In yet an additional embodiment, the Therapeutics of theinvention are delivered by way of a pump (see Langer, supra; Sefton, CRCCrit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507(1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

[1402] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[1403] For parenteral administration, in one embodiment, the Therapeuticis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

[1404] Generally, the formulations are prepared by contacting theTherapeutic uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

[1405] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[1406] The Therapeutic is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

[1407] Any pharmaceutical used for therapeutic administration can besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

[1408] Therapeutics ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

[1409] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the Therapeutics of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, theTherapeutics may be employed in conjunction with other therapeuticcompounds.

[1410] The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeuticsof the invention are administered in combination with alum. In anotherspecific embodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[1411] The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second. In one embodiment, the Therapeutics of theinvention are administered in combination with members of the TNFfamily. TNF, TNF-related or TNF-like molecules that may be administeredwith the Therapeutics of the invention include, but are not limited to,soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL,FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (InternationalPublication No. WO 96/14328), AIM-I (International Publication No. WO97/33899), endokine-alpha (International Publication No. WO 98/07880),TR6 (International Publication No. WO 98/30694), OPG, andneutrokine-alpha (International Publication No. WO 98/18921, OX40, andnerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3(International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO 98/30694), TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892), TR10 (International Publication No. WO98/54202), 312C2 (International Publication No. WO 98/06842), and TR12,and soluble forms CD154, CD70, and CD153.

[1412] In certain embodiments, Therapeutics of the invention areadministered in combination with antiretroviral agents, nucleosidereverse transcriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the Therapeuticsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith Therapeutics of the invention to treat AIDS and/or to prevent ortreat HIV infection.

[1413] In other embodiments, Therapeutics of the invention may beadministered in combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAME™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMINE™, and/or ATOVAQUONE™to prophylactically treat or prevent an opportunistic Pneumocystiscarinii pneumonia infection. In another specific embodiment,Therapeutics of the invention are used in any combination withISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™,and/or AZITHROMYCIN™ to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with PYRIMETHAMINE™ and/orLEUCOVORIN™ to prophylactically treat or prevent an opportunisticToxoplasma gondii infection. In another specific embodiment,Therapeutics of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

[1414] In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

[1415] In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

[1416] Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the Therapeutics of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

[1417] In specific embodiments, Therapeutics of the invention areadministered in combination with immunosuppressants. Immunosuppressantspreparations that may be administered with the Therapeutics of theinvention include, but are not limited to, ORTHOCLONE™ (OKT3),SANDIMMUNE™/NEORAL™/SANGDYA™ (cyclosporin), PROGRAF™ (tacrolimus),CELLCEPT™ (mycophenolate), Azathioprine, glucorticosteroids, andRAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants maybe used to prevent rejection of organ or bone marrow transplantation.

[1418] In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, Therapeutics of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

[1419] In an additional embodiment, the Therapeutics of the inventionare administered alone or in combination with an anti-inflammatoryagent. Anti-inflammatory agents that may be administered with theTherapeutics of the invention include, but are not limited to,glucocorticoids and the nonsteroidal anti-inflammatories,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

[1420] In another embodiment, compostions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the Therapeuticsof the invention include, but are not limited to, antibiotic derivatives(e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin);antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil,5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid,plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g.,carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[1421] In a specific embodiment, Therapeutics of the invention areadministered in combination with CHOP (cyclophosphamide, doxorubicin,vincristine, and prednisone) or any combination of the components ofCHOP. In another embodiment, Therapeutics of the invention areadministered in combination with Rituximab. In a further embodiment,Therapeutics of the invention are administered with Rituxmab and CHOP,or Rituxmab and any combination of the components of CHOP.

[1422] In an additional embodiment, the Therapeutics of the inventionare administered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

[1423] In an additional embodiment, the Therapeutics of the inventionare administered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PIGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PIGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are incorporated herein by reference herein.

[1424] In an additional embodiment, the Therapeutics of the inventionare administered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to, LEUKINE™(SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTIM™).

[1425] In an additional embodiment, the Therapeutics of the inventionare administered in combination with Fibroblast Growth Factors.Fibroblast Growth Factors that may be administered with the Therapeuticsof the invention include, but are not limited to, FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12,FGF-13, FGF-14, and FGF-15.

[1426] In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 24 Method of Treating Decreased Levels of the Polypeptide

[1427] The present invention relates to a method for treating anindividual in need of an increased level of a polypeptide of theinvention in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of an agonistof the invention (including polypeptides of the invention). Moreover, itwill be appreciated that conditions caused by a decrease in the standardor normal expression level of a secreted protein in an individual can betreated by administering the polypeptide of the present invention,preferably in the secreted form. Thus, the invention also provides amethod of treatment of an individual in need of an increased level ofthe polypeptide comprising administering to such an individual aTherapeutic comprising an amount of the polypeptide to increase theactivity level of the polypeptide in such an individual.

[1428] For example, a patient with decreased levels of a polypeptidereceives a daily dose 0.1-100 ug/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in the secreted form.The exact details of the dosing scheme, based on administration andformulation, are provided in Example 23.

Example 25 Method of Treating Increased Levels of the Polypeptide

[1429] The present invention also relates to a method of treating anindividual in need of a decreased level of a polypeptide of theinvention in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of anantagonist of the invention (including polypeptides and antibodies ofthe invention).

[1430] In one example, antisense technology is used to inhibitproduction of a polypeptide of the present invention. This technology isone example of a method of decreasing levels of a polypeptide,preferably a secreted form, due to a variety of etiologies, such ascancer. For example, a patient diagnosed with abnormally increasedlevels of a polypeptide is administered intravenously antisensepolynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days.This treatment is repeated after a 7-day rest period if the treatmentwas well tolerated. The formulation of the antisense polynucleotide isprovided in Example 23.

Example 26 Method of Treatment Using Gene Therapy-Ex Vivo

[1431] One method of gene therapy transplants fibroblasts, which arecapable of expressing a polypeptide, onto a patient. Generally,fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin) is added. The flasks are then incubated at 37 degreeC. for approximately one week.

[1432] At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

[1433] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flankedby the long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[1434] The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 1 using primers andhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HBB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

[1435] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[1436] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his. Once thefibroblasts have been efficiently infected, the fibroblasts are analyzedto determine whether protein is produced.

[1437] The engineered fibroblasts are then transplanted onto the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads.

Example 27 Gene Therapy Using Endosenous Genes Corresponding toPolynucleotides of the Invention

[1438] Another method of gene therapy according to the present inventioninvolves operably associating the endogenous polynucleotide sequence ofthe invention with a promoter via homologous recombination as described,for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired.

[1439] Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous polynucleotide sequence, flanking the promoter. Thetargeting sequence will be sufficiently near the 5′ end of thepolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination. The promoter and thetargeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

[1440] The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

[1441] In this Example, the polynucleotide constructs are administeredas naked polynucleotides via electroporation. However, thepolynucleotide constructs may also be administered withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, precipitating agents, etc. Such methods of delivery areknown in the art.

[1442] Once the cells are transfected, homologous recombination willtake place which results in the promoter being operably linked to theendogenous polynucleotide sequence. This results in the expression ofpolynucleotide corresponding to the polynucleotide in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

[1443] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in DMEM+10% fetal calf serum. Exponentiallygrowing or early stationary phase fibroblasts are trypsinized and rinsedfrom the plastic surface with nutrient medium. An aliquot of the cellsuspension is removed for counting, and the remaining cells aresubjected to centrifugation. The supernatant is aspirated and the pelletis resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3,137 mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO4, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

[1444] Plasmid DNA is prepared according to standard techniques. Forexample, to construct a plasmid for targeting to the locus correspondingto the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas,Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplifiedby PCR with an XbaI site on the 5′ end and a BamHI site on the 3′end.Two non-coding sequences are amplified via PCR: one non-coding sequence(fragment 1) is amplified with a HindIII site at the 5′ end and an Xbasite at the 3′end; the other non-coding sequence (fragment 2) isamplified with a BamHI site at the 5′end and a HindIII site at the3′end. The CMV promoter and the fragments (1 and 2) are digested withthe appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1-XbaI;fragment 2-BamHI) and ligated together. The resulting ligation productis digested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

[1445] Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrodegap (Bio-Rad). The final DNA concentration is generally at least 120μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5×10⁶cells) is then added to the cuvette, and the cell suspension and DNAsolutions are gently mixed. Electroporation is performed with aGene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960μF and 250-300 V, respectively. As voltage increases, cell survivaldecreases, but the percentage of surviving cells that stably incorporatethe introduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

[1446] Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

[1447] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product. Thefibroblasts can then be introduced into a patient as described above.

Example 28 Method of Treatment Using Gene Therapy—In Vivo

[1448] Another aspect of the present invention is using in vivo genetherapy methods to treat disorders, diseases and conditions. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) sequences into an animal to increase ordecrease the expression of the polypeptide. The polynucleotide of thepresent invention may be operatively linked to a promoter or any othergenetic elements necessary for the expression of the polypeptide by thetarget tissue. Such gene therapy and delivery techniques and methods areknown in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat.Nos. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res.35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997);Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., GeneTher. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290(1996) (incorporated herein by reference).

[1449] The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically acceptable liquid or aqueous carrier.

[1450] The term “naked” polynucleotide, DNA or RNA, refers to sequencesthat are free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the polynucleotides of the present invention may alsobe delivered in liposome formulations (such as those taught in FeignerP. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. etal. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods wellknown to those skilled in the art.

[1451] The polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication. Anystrong promoter known to those skilled in the art can be used fordriving the expression of DNA. Unlike other gene therapies techniques,one major advantage of introducing naked nucleic acid sequences intotarget cells is the transitory nature of the polynucleotide synthesis inthe cells. Studies have shown that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of up to six months.

[1452] The polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

[1453] For the naked polynucleotide injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 g/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

[1454] The dose response effects of injected polynucleotide in muscle invivo is determined as follows. Suitable template DNA for production ofmRNA coding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

[1455] Five to six week old female and male Balb/C mice are anesthetizedby intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cmincision is made on the anterior thigh, and the quadriceps muscle isdirectly visualized. The template DNA is injected in 0.1 ml of carrierin a 1 cc syringe through a 27 gauge needle over one minute,approximately 0.5 cm from the distal insertion site of the muscle intothe knee and about 0.2 cm deep. A suture is placed over the injectionsite for future localization, and the skin is closed with stainlesssteel clips.

[1456] After an appropriate incubation time (e.g., 7 days) muscleextracts are prepared by excising the entire quadriceps. Every fifth 15um cross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for protein expression maybe done in a similar fashion except that quadriceps from different miceare harvested at different times. Persistence of DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in humans andother animals using naked DNA.

Example 29 Transgenic Animals

[1457] The polypeptides of the invention can also be expressed intransgenic animals. Animals of any species, including, but not limitedto, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats,sheep, cows and non-human primates, e.g., baboons, monkeys, andchimpanzees may be used to generate transgenic animals. In a specificembodiment, techniques described herein or otherwise known in the art,are used to express polypeptides of the invention in humans, as part ofa gene therapy protocol.

[1458] Any technique known in the art may be used to introduce thetransgene (i.e., polynucleotides of the invention) into animals toproduce the founder lines of transgenic animals. Such techniquesinclude, but are not limited to, pronuclear microinjection (Paterson etal., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al.,Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology(NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191(1989)); retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)),blastocysts or embryos; gene targeting in embryonic stem cells (Thompsonet al., Cell 56:313-321 (1989)); electroporation of cells or embryos(Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of thepolynucleotides of the invention using a gene gun (see, e.g., Ulmer etal., Science 259:1745 (1993); introducing nucleic acid constructs intoembryonic pleuripotent stem cells and transferring the stem cells backinto the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,Cell 57:717-723 (1989); etc. For a review of such techniques, seeGordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989),which is incorporated by reference herein in its entirety.

[1459] Any technique known in the art may be used to produce transgenicclones containing polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

[1460] The present invention provides for transgenic animals that carrythe transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular-cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

[1461] Once transgenic animals have been generated, the expression ofthe recombinant gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

[1462] Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

[1463] Transgenic animals of the invention have uses which include, butare not limited to, animal model systems useful in elaborating thebiological function of polypeptides of the present invention, studyingconditions and/or disorders associated with aberrant expression, and inscreening for compounds effective in ameliorating such conditions and/ordisorders.

Example 30 Knock-Out Animals

[1464] Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

[1465] In further embodiments of the invention, cells that aregenetically engineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

[1466] Alternatively, the cells can be incorporated into a matrix andimplanted in the body, e.g., genetically engineered fibroblasts can beimplanted as part of a skin graft; genetically engineered endothelialcells can be implanted as part of a lymphatic or vascular graft. (See,for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan &Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated byreference herein in its entirety).

[1467] When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

[1468] Transgenic and “knock-out” animals of the invention have useswhich include, but are not limited to, animal model systems useful inelaborating the biological function of polypeptides of the presentinvention, studying conditions and/or disorders associated with aberrantexpression, and in screening for compounds effective in amelioratingsuch conditions and/or disorders.

Example 31 Production of an Antibody

[1469] Hybridoma Technology

[1470] The antibodies of the present invention can be prepared by avariety of methods. (See, Current Protocols, Chapter 2.) As one exampleof such methods, cells expressing XXX are administered to an animal toinduce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of XXX protein is prepared and purifiedto render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

[1471] Monoclonal antibodies specific for protein XXX are prepared usinghybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler etal., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol.6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal(preferably a mouse) is immunized with XXX polypeptide or, morepreferably, with a secreted XXX polypeptide-expressing cell. Suchpolypeptide-expressing cells are cultured in any suitable tissue culturemedium, preferably in Earle's modified Eagle's medium supplemented with10% fetal bovine serum (inactivated at about 56° C.), and supplementedwith about 10 g/l of nonessential amino acids, about 1,000 U/ml ofpenicillin, and about 100 μg/ml of streptomycin.

[1472] The splenocytes of such mice are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP20), available fromthe ATCC. After fusion, the resulting hybridoma cells are selectivelymaintained in HAT medium, and then cloned by limiting dilution asdescribed by Wands et al. (Gastroenterology 80:225-232 (1981)). Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding the XXXpolypeptide.

[1473] Alternatively, additional antibodies capable of binding to XXXpolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the XXXprotein-specific antibody can be blocked by XXX. Such antibodiescomprise anti-idiotypic antibodies to the XXX protein-specific antibodyand are used to immunize an animal to induce formation of further XXXprotein-specific antibodies.

[1474] For in vivo use of antibodies in humans, an antibody is“humanized”. Such antibodies can be produced using genetic constructsderived from hybridoma cells producing the monoclonal antibodiesdescribed above. Methods for producing chimeric and humanized antibodiesare known in the art and are discussed herein. (See, for review,Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533;Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984);Neuberger et al., Nature 314:268 (1985).)

[1475] b) Isolation of Antibody Fragments Directed Against XXX from aLibrary of scFvs

[1476] Naturally occurring V-genes isolated from human PBLs areconstructed into a library of antibody fragments which containreactivities against XXX to which the donor may or may not have beenexposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein byreference in its entirety).

[1477] Rescue of the Library. A library of scFvs is constructed from theRNA of human PBLs as described in PCT publication WO 92/01047. To rescuephage displaying antibody fragments, approximately 109 E. coli harboringthe phagemid are used to inoculate 50 ml of 2×TY containing 1% glucoseand 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8with shaking. Five ml of this culture is used to innoculate 50 ml of2×TY-AMP-GLU, 2×10⁸ TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 ug/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

[1478] M13 delta gene III is prepared as follows: M13 delta gene IIIhelper phage does not encode gene III protein, hence the phage(mid)displaying antibody fragments have a greater avidity of binding toantigen. Infectious M13 delta gene III particles are made by growing thehelper phage in cells harboring a pUC19 derivative supplying the wildtype gene III protein during phage morphogenesis. The culture isincubated for 1 hour at 37° C. without shaking and then for a furtherhour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μgampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight,shaking at 37° C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990),resuspended in 2 ml PBS and passed through a 0.45 μm filter (MinisartNML; Sartorius) to give a final concentration of approximately 1013transducing units/ml (ampicillin-resistant clones).

[1479] Panning of the Library. Immunotubes (Nunc) are coated overnightin PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

[1480] Characterization of Binders. Eluted phage from the 3rd and 4throunds of selection are used to infect E. coli HB 2151 and soluble scFvis produced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing. These ELISApositive clones may also be further characterized by techniques known inthe art, such as, for example, epitope mapping, binding affinity,receptor signal transduction, ability to block or competitively inhibitantibody/antigen binding, and competitive agonistic or antagonisticactivity.

Example 32 Assays Detecting Stimulation or Inhibition of B cellProliferation and Differentiation

[1481] Generation of functional humoral immune responses requires bothsoluble and cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

[1482] One of the best studied classes of B-cell co-stimulatory proteinsis the TNF-superfamily. Within this family CD40, CD27, and CD30 alongwith their respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

[1483] In Vitro Assay—Purified polypeptides of the invention, ortruncated forms thereof, is assessed for its ability to induceactivation, proliferation, differentiation or inhibition and/or death inB-cell populations and their precursors. The activity of thepolypeptides of the invention on purified human tonsillar B cells,measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, isassessed in a standard B-lymphocyte co-stimulation assay in whichpurified tonsillar B cells are cultured in the presence of eitherformalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilizedanti-human IgM antibody as the priming agent. Second signals such asIL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cellproliferation as measured by tritiated-thymidine incorporation. Novelsynergizing agents can be readily identified using this assay. The assayinvolves isolating human tonsillar B cells by magnetic bead (MACS)depletion of CD3-positive cells. The resulting cell population isgreater than 95% B cells as assessed by expression of CD45R(B220).

[1484] Various dilutions of each sample are placed into individual wellsof a 96-well plate to which are added 10⁵ B-cells suspended in culturemedium (RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100U/ml penicillin,10 ug/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of150 ul. Proliferation or inhibition is quantitated by a 20 h pulse (1uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

[1485] In Vivo Assay—BALB/c mice are injected (i.p.) twice per day withbuffer only, or 2 mg/Kg of a polypeptide of the invention, or truncatedforms thereof. Mice receive this treatment for 4 consecutive days, atwhich time they are sacrificed and various tissues and serum collectedfor analyses. Comparison of H&E sections from normal spleens and spleenstreated with polypeptides of the invention identify the results of theactivity of the polypeptides on spleen cells, such as the diffusion ofperi-arterial lymphatic sheaths, and/or significant increases in thenucleated cellularity of the red pulp regions, which may indicate theactivation of the differentiation and proliferation of B-cellpopulations. Immunohistochemical studies using a B cell marker,anti-CD45R(B220), are used to determine whether any physiologicalchanges to splenic cells, such as splenic disorganization, are due toincreased B-cell representation within loosely defined B-cell zones thatinfiltrate established T-cell regions.

[1486] Flow cytometric analyses of the spleens from mice treated withpolypeptide is used to indicate whether the polypeptide specificallyincreases the proportion of ThB+, CD45R(B220)dull B cells over thatwhich is observed in control mice.

[1487] Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andpolypeptide-treated mice.

[1488] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides of the invention (e.g., gene therapy), agonists, and/orantagonists of polynucleotides or polypeptides of the invention.

Example 33 T Cell Proliferation Assay

[1489] A CD3-induced proliferation assay is performed on PBMCs and ismeasured by the uptake of H-thymidine. The assay is performed asfollows. Ninety-six well plates are coated with 100 μl/well of mAb toCD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1) overnightat 4 degrees C. (1 μg/ml in 0.05M bicarbonate buffer, pH 9.5), thenwashed three times with PBS. PBMC are isolated by F/H gradientcentrifugation from human peripheral blood and added to quadruplicatewells (5×10⁴/well) of mAb coated plates in RPMI containing 10% FCS andP/S in the presence of varying concentrations of polypeptides of theinvention (total volume 200 ul). Relevant protein buffer and mediumalone are controls. After 48 hr. culture at 37 degrees C., plates arespun for 2 min. at 1000 rpm and 100 μl of supernatant is removed andstored −20 degrees C. for measurement of IL-2 (or other cytokines) ifeffect on proliferation is observed. Wells are supplemented with 100 ulof medium containing 0.5 uCi of ³H-thymidine and cultured at 37 degreesC. for 18-24 hr. Wells are harvested and incorporation of ³H-thymidineused as a measure of proliferation. Anti-CD3 alone is the positivecontrol for proliferation. IL-2 (100 U/ml) is also used as a controlwhich enhances proliferation. Control antibody which does not induceproliferation of T cells is used as the negative controls for theeffects of polypeptides of the invention.

[1490] The studies described in this example tested activity ofpolypeptides of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides of the invention (e.g., gene therapy), agonists, and/orantagonists of polynucleotides or polypeptides of the invention.

Example 34 Effect of Polypeptides of the Invention on the Expression ofMHC Class II, Costimulatory and Adhesion Molecules and CellDifferentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

[1491] Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-α,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFCγR11, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

[1492] FACS analysis of surface antigens is performed as follows. Cellsare treated 1-3 days with increasing concentrations of polypeptides ofthe invention or LPS (positive control), washed with PBS containing 1%BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution ofappropriate FITC− or PE-labeled monoclonal antibodies for 30 minutes at4 degrees C. After an additional wash, the labeled cells are analyzed byflow cytometry on a FACScan (Becton Dickinson).

[1493] Effect on the production of cytokines. Cytokines generated bydendritic cells, in particular IL-12, are important in the initiation ofT-cell dependent immune responses. IL-12 strongly influences thedevelopment of Th1 helper T-cell immune response, and induces cytotoxicT and NK cell function. An ELISA is used to measure the IL-12 release asfollows. Dendritic cells (106/ml) are treated with increasingconcentrations of polypeptides of the invention for 24 hours. LPS (100ng/ml) is added to the cell culture as positive control. Supernatantsfrom the cell cultures are then collected and analyzed for IL-12 contentusing commercial ELISA kit (e.g, R & D Systems (Minneapolis, Minn.)).The standard protocols provided with the kits are used.

[1494] Effect on the expression of MHC Class II, costimulatory andadhesion molecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

[1495] FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations ofpolypeptides of the invention or LPS (positive control), washed with PBScontaining 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20dilution of appropriate FITC— or PE-labeled monoclonal antibodies for 30minutes at 4 degrees c. After an additional wash, the labeled cells areanalyzed by flow cytometry on a FACScan (Becton Dickinson).

[1496] Monocyte activation and/or increased survival. Assays formolecules that activate (or alternatively, inactivate) monocytes and/orincrease monocyte survival (or alternatively, decrease monocytesurvival) are known in the art and may routinely be applied to determinewhether a molecule of the invention functions as an inhibitor oractivator of monocytes. Polypeptides, agonists, or antagonists of theinvention can be screened using the three assays described below. Foreach of these assays, Peripheral blood mononuclear cells (PBMC) arepurified from single donor leukopacks (American Red Cross, Baltimore,Md.) by centrifugation through a Histopaque gradient (Sigma). Monocytesare isolated from PBMC by counterflow centrifugal elutriation.

[1497] Monocyte Survival Assay. Human peripheral blood monocytesprogressively lose viability when cultured in absence of serum or otherstimuli. Their death results from internally regulated process(apoptosis). Addition to the culture of activating factors, such asTNF-alpha dramatically improves cell survival and prevents DNAfragmentation. Propidium iodide (PI) staining is used to measureapoptosis as follows. Monocytes are cultured for 48 hours inpolypropylene tubes in serum-free medium (positive control), in thepresence of 100 ng/ml TNF-alpha (negative control), and in the presenceof varying concentrations of the compound to be tested. Cells aresuspended at a concentration of 2×10⁶/ml in PBS containing PI at a finalconcentration of 5 μg/ml, and then incubaed at room temperature for 5minutes before FACScan analysis. PI uptake has been demonstrated tocorrelate with DNA fragmentation in this experimental paradigm.

[1498] Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×10⁵ cells/mlwith increasing concentrations of the a polypeptide of the invention andunder the same conditions, but in the absence of the polypeptide. ForIL-12 production, the cells are primed overnight with IFN (100 U/ml) inpresence of a polypeptide of the invention. LPS (10 ng/ml) is thenadded. Conditioned media are collected after 24 h and kept frozen untiluse. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performedusing a commercially available ELISA kit (e.g, R & D Systems(Minneapolis, Minn.)) and applying the standard protocols provided withthe kit.

[1499] Oxidative burst. Purified monocytes are plated in 96-w plate at2-1×10⁵ cell/well. Increasing concentrations of polypeptides of theinvention are added to the wells in a total volume of 0.2 ml culturemedium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3 daysincubation, the plates are centrifuged and the medium is removed fromthe wells. To the macrophage monolayers, 0.2 ml per well of phenol redsolution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mMdextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, togetherwith the stimulant (200 nM PMA). The plates are incubated at 37° C. for2 hours and the reaction is stopped by adding 20 μl 1N NaOH per well.The absorbance is read at 610 nm. To calculate the amount of H₂O₂produced by the macrophages, a standard curve of a H₂O₂ solution ofknown molarity is performed for each experiment.

[1500] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolypeptides, polynucleotides (e.g., gene therapy), agonists, and/orantagonists of the invention.

Example 35 Biological Effects of Polypeptides of the Invention

[1501] Astrocyte and Neuronal Assays

[1502] Recombinant polypeptides of the invention, expressed inEscherichia coli and purified as described above, can be tested foractivity in promoting the survival, neurite outgrowth, or phenotypicdifferentiation of cortical neuronal cells and for inducing theproliferation of glial fibrillary acidic protein immunopositive cells,astrocytes. The selection of cortical cells for the bioassay is based onthe prevalent expression of FGF-1 and FGF-2 in cortical structures andon the previously reported enhancement of cortical neuronal survivalresulting from FGF-2 treatment. A thymidine incorporation assay, forexample, can be used to elucidate a polypeptide of the invention'sactivity on these cells.

[1503] Moreover, previous reports describing the biological effects ofFGF-2 (basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of a polypeptideof the invention to induce neurite outgrowth can be compared to theresponse achieved with FGF-2 using, for example, a thymidineincorporation assay.

[1504] Fibroblast and Endothelial Cell Assays

[1505] Human lung fibroblasts are obtained from Clonetics (San Diego,Calif.) and maintained in growth media from Clonetics. Dermalmicrovascular endothelial cells are obtained from Cell Applications (SanDiego, Calif.). For proliferation assays, the human lung fibroblasts anddermal microvascular endothelial cells can be cultured at 5,000cells/well in a 96-well plate for one day in growth medium. The cellsare then incubated for one day in 0.1% BSA basal medium. After replacingthe medium with fresh 0.1% BSA medium, the cells are incubated with thetest proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento,Calif.) is added to each well to a final concentration of 10%. The cellsare incubated for 4 hr. Cell viability is measured by reading in aCytoFluor fluorescence reader. For the PGE₂ assays, the human lungfibroblasts are cultured at 5,000 cells/well in a 96-well plate for oneday. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or polypeptides of the invention with or withoutIL-1α for 24 hours. The supernatants are collected and assayed for PGE₂by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the humanlung fibroblasts are cultured at 5,000 cells/well in a 96-well plate forone day. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or with or without polypeptides of the inventionIL-1α for 24 hours. The supernatants are collected and assayed for IL-6by ELISA kit (Endogen, Cambridge, Mass.).

[1506] Human lung fibroblasts are cultured with FGF-2 or polypeptides ofthe invention for 3 days in basal medium before the addition of AlamarBlue to assess effects on growth of the fibroblasts. FGF-2 should show astimulation at 10-2500 ng/ml which can be used to compare stimulationwith polypeptides of the invention.

[1507] Parkinson Models.

[1508] The loss of motor function in Parkinson's disease is attributedto a deficiency of striatal dopamine resulting from the degeneration ofthe nigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP⁺) and released.Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP⁺ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

[1509] It has been demonstrated in tissue culture paradigms that FGF-2(basic FGF) has trophic activity towards nigral dopaminergic neurons(Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

[1510] Based on the data with FGF-2, polypeptides of the invention canbe evaluated to determine whether it has an action similar to that ofFGF-2 in enhancing dopaminergic neuronal survival in vitro and it canalso be tested in vivo for protection of dopaminergic neurons in thestriatum from the damage associated with MPTP treatment. The potentialeffect of a polypeptide of the invention is first examined in vitro in adopaminergic neuronal cell culture paradigm. The cultures are preparedby dissecting the midbrain floor plate from gestation day 14 Wistar ratembryos. The tissue is dissociated with trypsin and seeded at a densityof 200,000 cells/cm on polyorthinine-laminin coated glass coverslips.The cells are maintained in Dulbecco's Modified Eagle's medium and F12medium containing hormonal supplements (N1). The cultures are fixed withparaformaldehyde after 8 days in vitro and are processed for tyrosinehydroxylase, a specific marker for dopminergic neurons,immunohistochemical staining. Dissociated cell cultures are preparedfrom embryonic rats. The culture medium is changed every third day andthe factors are also added at that time.

[1511] Since the dopaminergic neurons are isolated from animals atgestation day 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if a polypeptide of the invention acts to prolong thesurvival of dopaminergic neurons, it would suggest that the polypeptidemay be involved in Parkinson's Disease.

[1512] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 36 The Effect of Polypeptides of the Invention on the Growth ofVascular Endothelial Cells

[1513] On day 1, human umbilical vein endothelial cells (HUVEC) areseeded at 2-5×10⁴ cells/35 mm dish density in M199 medium containing 4%fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/mlendothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day2, the medium is replaced with M199 containing 10% FBS, 8 units/mlheparin. A polypeptide having the amino acid sequence of SEQ ID NO:Y,and positive controls, such as VEGF and basic FGF (bFGF) are added, atvarying concentrations. On days 4 and 6, the medium is replaced. On day8, cell number is determined with a Coulter Counter.

[1514] An increase in the number of HUVEC cells indicates that thepolypeptide of the invention may proliferate vascular endothelial cells.

[1515] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 37 Stimulatory Effect of Polypeptides of the Invention on theProliferation of Vascular Endothelial Cells

[1516] For evaluation of mitogenic activity of growth factors, thecolorimetric MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)₂H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 mL serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF₁₆₅ or a polypeptide of the invention in 0.5%FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours.20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed toincubate for 1 hour at 37° C. before measuring the absorbance at 490 nmin an ELISA plate reader. Background absorbance from control wells (somemedia, no cells) is subtracted, and seven wells are performed inparallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol.30A:512-518 (1994).

[1517] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 38 Inhibition of PDGF-induced Vascular Smooth Muscle CellProliferation Stimulatory Effect

[1518] HAoSMC proliferation can be measured, for example, by BrdUrdincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then,the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h,immunocytochemistry is performed by using BrdUrd Staining Kit (ZymedLaboratories). In brief, the cells are incubated with the biotinylatedmouse anti-BrdUrd antibody at 4 degrees C. for 2 h after being exposedto denaturing solution and then incubated with thestreptavidin-peroxidase and diaminobenzidine. After counterstaining withhematoxylin, the cells are mounted for microscopic examination, and theBrdUrd-positive cells are counted. The BrdUrd index is calculated as apercent of the BrdUrd-positive cells to the total cell number. Inaddition, the simultaneous detection of the BrdUrd staining (nucleus)and the FITC uptake (cytoplasm) is performed for individual cells by theconcomitant use of bright field illumination and dark field-UVfluorescent illumination. See, Hayashida et al., J. Biol. Chem.6:271(36):21985-21992 (1996).

[1519] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 39 Stimulation of Endothelial Migration

[1520] This example will be used to explore the possibility that apolypeptide of the invention may stimulate lymphatic endothelial cellmigration.

[1521] Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, MD; Falk, W., etal., J. Immunological Methods 1980;33:239-247).Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um(Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for atleast 6 hours at room temperature and dried under sterile air. Testsubstances are diluted to appropriate concentrations in M199supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of thefinal dilution is placed in the lower chamber of the modified Boydenapparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures arewashed and trypsinized for the minimum time required to achieve celldetachment. After placing the filter between lower and upper chamber,2.5×10⁵ cells suspended in 50 ul M199 containing 1% FBS are seeded inthe upper compartment. The apparatus is then incubated for 5 hours at37° C. in a humidified chamber with 5% CO₂ to allow cell migration.After the incubation period, the filter is removed and the upper side ofthe filter with the non-migrated cells is scraped with a rubberpoliceman. The filters are fixed with methanol and stained with a Giemsasolution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration isquantified by counting cells of three random high-power fields (40×) ineach well, and all groups are performed in quadruplicate.

[1522] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 40 Stimulation of Nitric Oxide Production by Endothelial Cells

[1523] Nitric oxide released by the vascular endothelium is believed tobe a mediator of vascular endothelium relaxation. Thus, activity of apolypeptide of the invention can be assayed by determining nitric oxideproduction by endothelial cells in response to the polypeptide.

[1524] Nitric oxide is measured in 96-well plates of confluentmicrovascular endothelial cells after 24 hours starvation and asubsequent 4 hr exposure to various levels of a positive control (suchas VEGF-1) and the polypeptide of the invention. Nitric oxide in themedium is determined by use of the Griess reagent to measure totalnitrite after reduction of nitric oxide-derived nitrate by nitratereductase. The effect of the polypeptide of the invention on nitricoxide release is examined on HUVEC.

[1525] Briefly, NO release from cultured HUVEC monolayer is measuredwith a NO-specific polarographic electrode connected to a NO meter(Iso-NO, World Precision Instruments Inc.) (1049). Calibration of the NOelements is performed according to the following equation:

2KNO₂+2 KI+2H₂SO₄6 2 NO+I₂+2H₂O+2K₂SO₄

[1526] The standard calibration curve is obtained by adding gradedconcentrations of KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing KI and H₂SO₄. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas (1050). The culture medium is removed and HUVECsare washed twice with Dulbecco's phosphate buffered saline. The cellsare then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates are kept on a slide warmer (Lab LineInstruments Inc.) To maintain the temperature at 37° C. The NO sensorprobe is inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) is used as apositive control. The amount of released NO is expressed as picomolesper 1×10⁶ endothelial cells. All values reported are means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

[1527] The studies described in this example tested activity ofpolypeptides of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 41 Effect of Polypepides of the Invention on Cord Formation inAngiogenesis

[1528] Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

[1529] CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications' CADMEC Growth Medium and used at passage 5. Forthe in vitro angiogenesis assay, the wells of a 48-well cell cultureplate are coated with Cell Applications' Attachment Factor Medium (200ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wellsat 7,500 cells/well and cultured overnight in Growth Medium. The GrowthMedium is then replaced with 300 mg Cell Applications' Chord FormationMedium containing control buffer or a polypeptide of the invention (0.1to 100 ng/ml) and the cells are cultured for an additional 48 hr. Thenumbers and lengths of the capillary-like chords are quantitated throughuse of the Boeckeler VIA-170 video image analyzer. All assays are donein triplicate.

[1530] Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.b-esteradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

[1531] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 42 Angiogenic Effect on Chick Chorioallantoic Membrane

[1532] Chick chorioallantoic membrane (CAM) is a well-established systemto examine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of polypeptides of the invention tostimulate angiogenesis in CAM can be examined.

[1533] Fertilized eggs of the White Leghorn chick (Gallus gallus) andthe Japanese qual (Coturnix coturnix) are incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old qualembryos is studied with the following methods.

[1534] On Day 4 of development, a window is made into the egg shell ofchick eggs. The embryos are checked for normal development and the eggssealed with cellotape. They are further incubated until Day 13.Thermanox coverslips (Nunc, Naperville, Ill.) are cut into disks ofabout 5 mm in diameter. Sterile and salt-free growth factors aredissolved in distilled water and about 3.3 mg/5 ml are pipetted on thedisks. After air-drying, the inverted disks are applied on CAM. After 3days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehydeand rinsed in 0.12 M sodium cacodylate buffer. They are photographedwith a stereo microscope [Wild M8] and embedded for semi- and ultrathinsectioning as described above. Controls are performed with carrier disksalone.

[1535] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 43 Angiogenesis Assay Using a Matrigel Implant in Mouse

[1536] In vivo angiogenesis assay of a polypeptide of the inventionmeasures the ability of an existing capillary network to form newvessels in an implanted capsule of murine extracellular matrix material(Matrigel). The protein is mixed with the liquid Matrigel at 4 degree C.and the mixture is then injected subcutaneously in mice where itsolidifies. After 7 days, the solid “plug” of Matrigel is removed andexamined for the presence of new blood vessels. Matrigel is purchasedfrom Becton Dickinson Labware/Collaborative Biomedical Products.

[1537] When thawed at 4 degree C. the Matrigel material is a liquid. TheMatrigel is mixed with a polypeptide of the invention at 150 ng/ml at 4degrees C. and drawn into cold 3 ml syringes. Female C57BI/6 miceapproximately 8 weeks old are injected with the mixture of Matrigel andexperimental protein at 2 sites at the midventral aspect of the abdomen(0.5 ml/site). After 7 days, the mice are sacrificed by cervicaldislocation, the Matrigel plugs are removed and cleaned (i.e., allclinging membranes and fibrous tissue is removed). Replicate whole plugsare fixed in neutral buffered 10% formaldehyde, embedded in paraffin andused to produce sections for histological examination after stainingwith Masson's Trichrome. Cross sections from 3 different regions of eachplug are processed. Selected sections are stained for the presence ofvWF. The positive control for this assay is bovine basic FGF (150ng/ml). Matrigel alone is used to determine basal levels ofangiogenesis.

[1538] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 44 Rescue of Ischemia in Rabbit Lower Limb Model

[1539] To study the in vivo effects of polynucleotides and polypeptidesof the invention on ischemia, a rabbit hindlimb ischemia model iscreated by surgical removal of one femoral arteries as describedpreviously (Takeshita et al., Am J. Pathol 147:1649-1660 (1995)). Theexcision of the femoral artery results in retrograde propagation ofthrombus and occlusion of the external iliac artery. Consequently, bloodflow to the ischemic limb is dependent upon collateral vesselsoriginating from the internal iliac artery (Takeshita et al. Am J.Pathol 147:1649-1660 (1995)). An interval of 10 days is allowed forpost-operative recovery of rabbits and development of endogenouscollateral vessels. At 10 day post-operatively (day 0), after performinga baseline angiogram, the internal iliac artery of the ischemic limb istransfected with 500 mg naked expression plasmid containing apolynucleotide of the invention by arterial gene transfer technologyusing a hydrogel-coated balloon catheter as described (Riessen et al.Hum Gene Ther. 4:749-758 (1993); Leclerc et al. J. Clin. Invest. 90:936-944 (1992)). When a polypeptide of the invention is used in thetreatment, a single bolus of 500 mg polypeptide of the invention orcontrol is delivered into the internal iliac artery of the ischemic limbover a period of 1 min. through an infusion catheter. On day 30, variousparameters are measured in these rabbits: (a) BP ratio—The bloodpressure ratio of systolic pressure of the ischemic limb to that ofnormal limb; (b) Blood Flow and Flow Reserve—Resting FL: the blood flowduring undilated condition and Max FL: the blood flow during fullydilated condition (also an indirect measure of the blood vessel amount)and Flow Reserve is reflected by the ratio of max FL: resting FL; (c)Angiographic Score—This is measured by the angiogram of collateralvessels. A score is determined by the percentage of circles in anoverlaying grid that with crossing opacified arteries divided by thetotal number m the rabbit thigh; (d) Capillary density—The number ofcollateral capillaries determined in light microscopic sections takenfrom hindlimbs.

[1540] The studies described in this example tested activity ofpolynucleotides and polypeptides of the invention. However, one skilledin the art could easily modify the exemplified studies to test theagonists, and/or antagonists of the invention.

Example 45 Effect of Polypeptides of the Invention on Vasodilation

[1541] Since dilation of vascular endothelium is important in reducingblood pressure, the ability of polypeptides of the invention to affectthe blood pressure in spontaneously hypertensive rats (SHR) is examined.Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of thepolypeptides of the invention are administered to 13-14 week oldspontaneously hypertensive rats (SHR). Data are expressed as the mean+/−SEM. Statistical analysis are performed with a paired t-test andstatistical significance is defined as p<0.05 vs. the response to bufferalone.

[1542] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 46 Rat Ischemic Skin Flap Model

[1543] The evaluation parameters include skin blood flow, skintemperature, and factor VIII immunohistochemistry or endothelialalkaline phosphatase reaction. Expression of polypeptides of theinvention, during the skin ischemia, is studied using in situhybridization.

[1544] The study in this model is divided into three parts as follows:

[1545] a) Ischemic skin

[1546] b) Ischemic skin wounds

[1547] c) Normal wounds

[1548] The experimental protocol includes:

[1549] a) Raising a 3×4 cm, single pedicle full-thickness random skinflap (myocutaneous flap over the lower back of the animal).

[1550] b) An excisional wounding (4-6 mm in diameter) in the ischemicskin (skin-flap).

[1551] c) Topical treatment with a polypeptide of the invention of theexcisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the followingvarious dosage ranges: 1 mg to 100 mg.

[1552] d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21post-wounding for histological, immunohistochemical, and in situstudies.

[1553] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 47 Peripheral Arterial Disease Model

[1554] Angiogenic therapy using a polypeptide of the invention is anovel therapeutic strategy to obtain restoration of blood flow aroundthe ischemia in case of peripheral arterial diseases. The experimentalprotocol includes:

[1555] a) One side of the femoral artery is ligated to create ischemicmuscle of the hindlimb, the other side of hindlimb serves as a control.

[1556] b) a polypeptide of the invention, in a dosage range of 20 mg-500mg, is delivered intravenously and/or intramuscularly 3 times (perhapsmore) per week for 2-3 weeks.

[1557] c) The ischemic muscle tissue is collected after ligation of thefemoral artery at 1, 2, and 3 weeks for the analysis of expression of apolypeptide of the invention and histology. Biopsy is also performed onthe other side of normal muscle of the contralateral hindlimb.

[1558] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 48 Ischemic Myocardial Disease Model

[1559] A polypeptide of the invention is evaluated as a potent mitogencapable of stimulating the development of collateral vessels, andrestructuring new vessels after coronary artery occlusion. Alteration ofexpression of the polypeptide is investigated in situ. The experimentalprotocol includes:

[1560] a) The heart is exposed through a left-side thoracotomy in therat. Immediately, the left coronary artery is occluded with a thinsuture (6-0) and the thorax is closed.

[1561] b) a polypeptide of the invention, in a dosage range of 20 mg-500mg, is delivered intravenously and/or intramuscularly 3 times (perhapsmore) per week for 24 weeks.

[1562] c) Thirty days after the surgery, the heart is removed andcross-sectioned for morphometric and in situ analyzes.

[1563] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 49 Rat Corneal Wound Healing Model

[1564] This animal model shows the effect of a polypeptide of theinvention on neovascularization. The experimental protocol includes:

[1565] a) Making a 1-1.5 mm long incision from the center of cornea intothe stromal layer.

[1566] b) Inserting a spatula below the lip of the incision facing theouter corner of the eye.

[1567] c) Making a pocket (its base is 1-1.5 mm form the edge of theeye).

[1568] d) Positioning a pellet, containing 50 ng-5 ug of a polypeptideof the invention, within the pocket.

[1569] e) Treatment with a polypeptide of the invention can also beapplied topically to the corneal wounds in a dosage range of 20 mg-500mg (daily treatment for five days).

[1570] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 50 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

[1571] Diabetic db+/db+Mouse ModeL

[1572] To demonstrate that a polypeptide of the invention acceleratesthe healing process, the genetically diabetic mouse model of woundhealing is used. The full thickness wound healing model in thedb+/db+mouse is a well characterized, clinically relevant andreproducible model of impaired wound healing. Healing of the diabeticwound is dependent on formation of granulation tissue andre-epithelialization rather than contraction (Gartner, M. H. et al., J.Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol.136:1235 (1990)).

[1573] The diabetic animals have many of the characteristic featuresobserved in Type II diabetes mellitus. Homozygous (db+/db+) mice areobese in comparison to their normal heterozygous (db+/+m) littermates.Mutant diabetic (db+/db+) mice have a single autosomal recessivemutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci.USA 77:283-293 (1982)). Animals show polyphagia, polydipsia andpolyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose,increased or normal insulin levels, and suppressed cell-mediatedimmunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M.et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. ofPathol. 114:46-55 (1985)). Peripheral neuropathy, myocardialcomplications, and microvascular lesions, basement membrane thickeningand glomerular filtration abnormalities have been described in theseanimals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertsonet al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest.40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6 (1982)).These homozygous diabetic mice develop hyperglycemia that is resistantto insulin analogous to human type II diabetes (Mandel et al., J.Immunol. 120:1375-1377 (1978)).

[1574] The characteristics observed in these animals suggests thathealing in this model may be similar to the healing observed in humandiabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

[1575] Genetically diabetic female C57BLJKsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

[1576] Wounding protocol is performed according to previously reportedmethods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

[1577] Wounds are visually examined and photographed at a fixed distanceat the day of surgery and at two day intervals thereafter. Wound closureis determined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

[1578] A polypeptide of the invention is administered using at a rangedifferent doses, from 4 mg to 500 mg per wound per day for 8 days invehicle. Vehicle control groups received 50 mL of vehicle solution.

[1579] Animals are euthanized on day 8 with an intraperitoneal injectionof sodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

[1580] Three groups of 10 animals each (5 diabetic and 5 non-diabeticcontrols) are evaluated: 1) Vehicle placebo control, 2) untreated group,and 3) treated group.

[1581] Wound closure is analyzed by measuring the area in the verticaland horizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64 mm², the corresponding size of the dermalpunch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

[1582] Specimens are fixed in 10% buffered formalin and paraffinembedded blocks are sectioned perpendicular to the wound surface (5 mm)and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with a polypeptide of the invention. This assessment includedverification of the presence of cell accumulation, inflammatory cells,capillaries, fibroblasts, re-epithelialization and epidermal maturity(Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibratedlens micrometer is used by a blinded observer.

[1583] Tissue sections are also stained immunohistochemically with apolyclonal rabbit anti-human keratin antibody using ABC Elite detectionsystem. Human skin is used as a positive tissue control while non-immuneIgG is used as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

[1584] Proliferating cell nuclear antigen/cyclin (PCNA) in skinspecimens is demonstrated by using anti-PCNA antibody (1:50) with an ABCElite detection system. Human colon cancer can serve as a positivetissue control and human brain tissue can be used as a negative tissuecontrol. Each specimen includes a section with omission of the primaryantibody and substitution with non-immune mouse IgG. Ranking of thesesections is based on the extent of proliferation on a scale of 0-8, thelower side of the scale reflecting slight proliferation to the higherside reflecting intense proliferation.

[1585] Experimental data are analyzed using an unpaired t test. A pvalue of <0.05 is considered significant.

[1586] B. Steroid Impaired Rat Model

[1587] The inhibition of wound healing by steroids has been welldocumented in various in vitro and in vivo systems (Wahl,Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action:Basic and Clinical Aspects. 280-302 (1989); Wahl et al., J. Immunol.115: 476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)).Glucocorticoids retard wound healing by inhibiting angiogenesis,decreasing vascular permeability (Ebert et al., An. Intern. Med.37:701-705 (1952)), fibroblast proliferation, and collagen synthesis(Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin.Invest. 61: 703-797 (1978)) and producing a transient reduction ofcirculating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797(1978); Wahl, “Glucocorticoids and wound healing”, In: AntiinflammatorySteroid Action: Basic and Clinical Aspects, Academic Press, New York,pp. 280-302 (1989)). The systemic administration of steroids to impairedwound healing is a well establish phenomenon in rats (Beck et al.,Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61:703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In:Antiinflammatory Steroid Action: Basic and Clinical Aspects, AcademicPress, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad.Sci. USA 86: 2229-2233 (1989)).

[1588] To demonstrate that a polypeptide of the invention can acceleratethe healing process, the effects of multiple topical applications of thepolypeptide on full thickness excisional skin wounds in rats in whichhealing has been impaired by the systemic administration ofmethylprednisolone is assessed.

[1589] Young adult male Sprague Dawley rats weighing 250-300 g (CharlesRiver Laboratories) are used in this example. The animals are purchasedat 8 weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

[1590] The wounding protocol is followed according to section A, above.On the day of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

[1591] Wounds are visually examined and photographed at a fixed distanceat the day of wounding and at the end of treatment. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

[1592] The polypeptide of the invention is administered using at a rangedifferent doses, from 4 mg to 500 mg per wound per day for 8 days invehicle. Vehicle control groups received 50 mL of vehicle solution.

[1593] Animals are euthanized on day 8 with an intraperitoneal injectionof sodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

[1594] Four groups of 10 animals each (5 with methylprednisolone and 5without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicleplacebo control 3) treated groups.

[1595] Wound closure is analyzed by measuring the area in the verticaland horizontal axis and obtaining the total area of the wound. Closureis then estimated by establishing the differences between the initialwound area (day 0) and that of post treatment (day 8). The wound area onday 1 is 64 mm², the corresponding size of the dermal punch.Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

[1596] Specimens are fixed in 10% buffered formalin and paraffinembedded blocks are sectioned perpendicular to the wound surface (5 mm)and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds allows assessment of whether the healingprocess and the morphologic appearance of the repaired skin is improvedby treatment with a polypeptide of the invention. A calibrated lensmicrometer is used by a blinded observer to determine the distance ofthe wound gap.

[1597] Experimental data are analyzed using an unpaired t test. A pvalue of <0.05 is considered significant.

[1598] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 51 Lymphadema Animal Model

[1599] The purpose of this experimental approach is to create anappropriate and consistent lymphedema model for testing the therapeuticeffects of a polypeptide of the invention in lymphangiogenesis andre-establishment of the lymphatic circulatory system in the rat hindlimb. Effectiveness is measured by swelling volume of the affected limb,quantification of the amount of lymphatic vasculature, total bloodplasma protein, and histopathology. Acute lymphedema is observed for7-10 days. Perhaps more importantly, the chronic progress of the edemais followed for up to 3-4 weeks.

[1600] Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately ˜350 g aredosed with Pentobarbital. Subsequently, the right legs are shaved fromknee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH.Blood is drawn for serum total protein testing. Circumference andvolumetric measurements are made prior to injecting dye into paws aftermarking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsalpaw). The intradermal dorsum of both right and left paws are injectedwith 0.05 ml of 1% Evan's Blue. Circumference and volumetricmeasurements are then made following injection of dye into paws.

[1601] Using the knee joint as a landmark, a mid-leg inguinal incisionis made circumferentially allowing the femoral vessels to be located.Forceps and hemostats are used to dissect and separate the skin flaps.After locating the femoral vessels, the lymphatic vessel that runs alongside and underneath the vessel(s) is located. The main lymphatic vesselsin this area are then electrically coagulated suture ligated.

[1602] Using a microscope, muscles in back of the leg (near thesemitendinosis and adductors) are bluntly dissected. The popliteal lymphnode is then located. The 2 proximal and 2 distal lymphatic vessels anddistal blood supply of the popliteal node are then and ligated bysuturing. The popliteal lymph node, and any accompanying adipose tissue,is then removed by cutting connective tissues.

[1603] Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (AJ Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of ˜0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

[1604] To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw before operation and daily for 7 days are measured. The effectplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

[1605] Circumference Measurements: Under brief gas anesthetic to preventlimb movement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople then those 2 readings are averaged. Readings are taken from bothcontrol and edematous limbs.

[1606] Volumetric Measurements: On the day of surgery, animals areanesthetized with Pentobarbital and are tested prior to surgery. Fordaily volumetrics animals are under brief halothane anesthetic (rapidimmobilization and quick recovery), both legs are shaved and equallymarked using waterproof marker on legs. Legs are first dipped in water,then dipped into instrument to each marked level then measured by Buxcoedema software(Chen/Victor). Data is recorded by one person, while theother is dipping the limb to marked area.

[1607] Blood-plasma protein measurements: Blood is drawn, spun, andserum separated prior to surgery and then at conclusion for totalprotein and Ca2+ comparison.

[1608] Limb Weight Comparison: After drawing blood, the animal isprepared for tissue collection. The limbs are amputated using aquillitine, then both experimental and control legs are cut at theligature and weighed. A second weighing is done as the tibio-cacanealjoint is disarticulated and the foot is weighed.

[1609] Histological Preparations: The transverse muscle located behindthe knee (popliteal) area is dissected and arranged in a metal mold,filled with freezeGel, dipped into cold methylbutane, placed intolabeled sample bags at −80EC until sectioning. Upon sectioning, themuscle is observed under fluorescent microscopy for lymphatics.

[1610] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 52 Suppression of TNF alpha-induced adhesion molecule expressionby a Polypeptide of the Invention

[1611] The recruitment of lymphocytes to areas of inflammation andangiogenesis involves specific receptor-ligand interactions between cellsurface adhesion molecules (CAMs) on lymphocytes and the vascularendothelium. The adhesion process, in both normal and pathologicalsettings, follows a multi-step cascade that involves intercellularadhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1(VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin)expression on endothelial cells (EC). The expression of these moleculesand others on the vascular endothelium determines the efficiency withwhich leukocytes may adhere to the local vasculature and extravasateinto the local tissue during the development of an inflammatoryresponse. The local concentration of cytokines and growth factorparticipate in the modulation of the expression of these CAMs.

[1612] Tumor necrosis factor alpha (TNF-a), a potent proinflammatorycytokine, is a stimulator of all three CAMs on endothelial cells and maybe involved in a wide variety of inflammatory responses, often resultingin a pathological outcome.

[1613] The potential of a polypeptide of the invention to mediate asuppression of TNF-a induced CAM expression can be examined. A modifiedELISA assay which uses ECs as a solid phase absorbent is employed tomeasure the amount of CAM expression on TNF-a treated ECs whenco-stimulated with a member of the FGF family of proteins.

[1614] To perform the experiment, human umbilical vein endothelial cell(HUVEC) cultures are obtained from pooled cord harvests and maintainedin growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidifiedincubator containing 5% CO₂. HUVECs are seeded in 96-well plates atconcentrations of 1×10⁴ cells/well in EGM medium at 37 degree C. for18-24 hrs or until confluent. The monolayers are subsequently washed 3times with a serum-free solution of RPMI-1640 supplemented with 100 U/mlpenicillin and 100 mg/ml streptomycin, and treated with a given cytokineand/or growth factor(s) for 24 h at 37 degree C. Following incubation,the cells are then evaluated for CAM expression.

[1615] Human Umbilical Vein Endothelial cells (HUVECs) are grown in astandard 96 well plate to confluence. Growth medium is removed from thecells and replaced with 90 ul of 199 Medium (10% FBS). Samples fortesting and positive or negative controls are added to the plate intriplicate (in 10 ul volumes). Plates are incubated at 37 degree C. foreither 5 h (selectin and integrin expression) or 24 h (integrinexpression only). Plates are aspirated to remove medium and 1001 μl of0.1% paraformaldehyde-PBS(with Ca++ and Mg++) is added to each well.Plates are held at 4° C. for 30 min.

[1616] Fixative is then removed from the wells and wells are washed 1×with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry.Add 10 μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA.

[1617] Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphotase(1:5,000 dilution) to each well and incubated at 37° C. for 30 min.Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-NitrophenolPhosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μlof pNPP substrate in glycine buffer is added to each test well. Standardwells in triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000 (10⁰)>10¹⁰^(−0.5)>10⁻¹>10⁻¹. 5 μl of each dilution is added to triplicate wellsand the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng,0.18 ng. 100 μl of pNNP reagent must then be added to each of thestandard wells. The plate must be incubated at 37° C. for 4 h. A volumeof 50 μl of 3M NaOH is added to all wells. The results are quantified ona plate reader at 405 nm. The background subtraction option is used onblank wells filled with glycine buffer only. The template is set up toindicate the concentration of AP-conjugate in each standard well [5.50ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of boundAP-conjugate in each sample.

[1618] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

[1619] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples. Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[1620] The entire disclosure of each document cited (including patents,patent applications, journal articles, abstracts, laboratory manuals,books, or other disclosures) in the Background of the Invention,Detailed Description, and Examples is hereby incorporated herein byreference. Further, the hard copy of the sequence listing submittedherewith and the corresponding computer readable form are bothincorporated herein by reference in their entireties.

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20040146930). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

What is claimed is:
 1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a polynucleotide fragment of SEQ ID NO:40 or a polynucleotide fragment of the cDNA sequence included in ATCC Deposit No:209782, which is hybridizable to SEQ ID NO:40; (b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:161 or a polypeptide fragment encoded by the cDNA sequence included in ATCC Deposit No:209782, which is hybridizable to SEQ ID NO:40; (c) a polynucleotide encoding a polypeptide domain of SEQ ID NO:161 or a polypeptide domain encoded by the cDNA sequence included in ATCC Deposit No:209782, which is hybridizable to SEQ ID NO:40; (d) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:161 or a polypeptide epitope encoded by the cDNA sequence included in ATCC Deposit No:209782, which is hybridizable to SEQ ID NO:40; (e) a polynucleotide encoding a polypeptide of SEQ ID NO:161 or the cDNA sequence included in ATCC Deposit No:209782, which is hybridizable to SEQ ID NO:40, having biological activity; (f) a polynucleotide which is a variant of SEQ ID NO:40; (g) a polynucleotide which is an allelic variant of SEQ ID NO:40; (h) a polynucleotide which encodes a species homologue of the SEQ ID NO:161; (i) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.
 2. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a secreted protein.
 3. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:161 or the polypeptide encoded by the cDNA sequence included in ATCC Deposit No:209782, which is hybridizable to SEQ ID NO:40.
 4. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO:40 or the cDNA sequence included in ATCC Deposit No:209782, which is hybridizable to SEQ ID NO:40.
 5. The isolated nucleic acid molecule of claim 2, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
 6. The isolated nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
 7. A recombinant vector comprising the isolated nucleic acid molecule of claim
 1. 8. A method of making a recombinant host cell comprising the isolated nucleic acid molecule of claim
 1. 9. A recombinant host cell produced by the method of claim
 8. 10. The recombinant host cell of claim 9 comprising vector sequences.
 11. An isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: (a) a polypeptide fragment of SEQ ID NO:161 or the encoded sequence included in ATCC Deposit No:209782; (b) a polypeptide fragment of SEQ ID NO:161 or the encoded sequence included in ATCC Deposit No:209782, having biological activity; (c) a polypeptide domain of SEQ ID NO:161 or the encoded sequence included in ATCC Deposit No:209782; (d) a polypeptide epitope of SEQ ID NO:161 or the encoded sequence included in ATCC Deposit No:209782; (e) a secreted form of SEQ ID NO:161 or the encoded sequence included in ATCC Deposit No:209782; (f) a full length protein of SEQ ID NO:161 or the encoded sequence included in ATCC Deposit No:209782; (g) a variant of SEQ ID NO:161; (h) an allelic variant of SEQ ID NO:161; or (i) a species homologue of the SEQ ID NO:161.
 12. The isolated polypeptide of claim 11, wherein the secreted form or the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.
 13. An isolated antibody that binds specifically to the isolated polypeptide of claim
 11. 14. A recombinant host cell that expresses the isolated polypeptide of claim
 11. 15. A method of making an isolated polypeptide comprising: (a) culturing the recombinant host cell of claim 14 under conditions such that said polypeptide is expressed; and (b) recovering said polypeptide.
 16. The polypeptide produced by claim
 15. 17. A method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polynucleotide of claim
 1. 18. A method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim
 11. 19. A method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the antibody of claim
 13. 20. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising: (a) determining the presence or absence of a mutation in the polynucleotide of claim 1; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.
 21. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising: (a) determining the presence or amount of expression of the polypeptide of claim 11 in a biological sample; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.
 22. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising: (a) using the antibody of claim 13 to determine the presence or amount of expression of a polypeptide that specifically binds said antibody; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.
 23. A method for identifying a binding partner to the polypeptide of claim 11 comprising: (a) contacting the polypeptide of claim 11 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide.
 24. The gene corresponding to the cDNA sequence encoding SEQ ID NO:161.
 25. A method of identifying an activity in a biological assay, wherein the method comprises: (a) expressing SEQ ID NO:40 in a cell; (b) isolating the supernatant; (c) detecting an activity in a biological assay; and (d) identifying the protein in the supernatant having the activity.
 26. The product produced by the method of claim
 23. 